HK1081863A - Methods for treating muscle injuries - Google Patents

Description

Methods for treating muscle damage

Background

The present invention relates to methods for treating muscle damage. In particular, the invention relates to methods of treating damaged muscles by administering a neurotoxin to the damaged muscles.

Muscle injuries include acute injuries to skeletal muscle such as contusions (bruises), lacerations, ischemia, sprains, and total ruptures. These injuries can cause great pain and can incapacitate the patient, rendering it inoperable or even incapable of normal daily activities. Among the acute injuries of skeletal muscle, sprains (also known as injuries resulting from stretching) are the most common. For example, sprains account for up to 30% of all injuries treated by professional or sports medical professionals. Garrett et al, Am J Sports Med, 24 (6): S2-S8, 1996.

Muscle spraining injuries are characterized by the destruction of the myo-tendon unit. The destruction of the myo-tendon unit can occur in any part of the muscle. This type of injury most commonly occurs near the tendon junctions (MTJs) of the superficial muscles that run around two joints, such as the rectus femoris, semitendinosus and gastrocnemius muscles.

Muscle sprains can result from erratic motion, or abnormal application of muscle. For example, centrifugal contraction uses fewer active motion units to generate higher forces. In such cases, during stretching, the over-extended muscle units experience excessive tension. The excessive tension can cause microscopic damage to the muscle contraction components, with the concentration appearing as random disruptions of the Z-line. When the muscles are damaged, the patient may experience a delayed onset muscular distress characterized by pain, weakness, and a limited range of motion. The pain is most intense about 1 to 2 days after muscle injury, and a limited range of motion may last for a week or more. More serious injuries may occur if a mild sprain of skeletal muscle is improperly treated.

Based on the severity of the injury and the nature of the hematoma, there are three categories of muscle sprains: (1) mild (one degree) sprain; a few muscle fibers torn; slight swelling and discomfort with no or minimal loss of strength and limited movement; (2) moderate (second degree) sprain; greater damage to muscle fibers with significant loss of strength, and (3) severe (three degree) sprains; tears across the entire muscle abdomen, resulting in total loss of muscle function.

Tearing of the intramuscular blood vessels during muscle sprains can often lead to a large number of hematomas. There are two different types of hematomas in damaged muscle: intramuscular and intermuscular hematomas. The first type, intramuscular hematomas, is limited in scale to the intact muscle fascia. Wherein extravasation of blood increases intramuscular pressure, compressing and limiting the size of the hematoma. Such hematomas produce pain and cause muscle loss of function. The second type, an internuscular hematoma, is formed when the muscle fascia breaks, and extravasated blood spreads into the internuscular space without significantly increasing the pressure within the muscle. This type of hematoma does not cause significant pain if the pressure in the muscle is not increased.

For the treatment of sprain injuries, it is critical to immobilize the injured muscle, especially within the first two to three days after injury, because the immediate movement of the injured muscle after injury often results in re-rupture of the original site of injury. Re-rupture can lead to more severe injury, delay healing and scar tissue. Jarvinen et al, Curr Opin Rheumatotol, Vol 12: 155-161(2000).

Re-rupture of the damaged area can be avoided by fixing the damaged muscle, preferably immediately after the injury. The fixation may allow the newly formed granulation tissue to gain sufficient tension to resist the force created by the contractible muscles.

One known method for immobilizing damaged/sprained muscles requires the use of physical restraints or tubes. For example, a collar may be used to immobilize flexors or extensors of a damaged neck. However, the application of restraints is often cumbersome and uncomfortable. Furthermore, for the damage of certain muscle groups, it is impractical or impossible to use physical restraints. For example, it is difficult to use a restraint to immobilize the sprained upper trapezius or maximum gluteus.

Botulinum toxin

The anaerobic, gram-positive bacterium clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which can cause a neuroparalytic illness in humans and animals called botulism. The spores of clostridium botulinum are found in soil and can grow in home canned food containers that are improperly sterilized and sealed, which is the cause of many botulism events. The effects of botulism typically manifest themselves 18 to 36 hours after consumption of a food product infected with a culture or spore of clostridium botulinum. The botulinum toxin apparently passes unattenuated through the lining of the gut and attacks peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.

For humans, botulinum toxin type A ("BoNT/A") is the most lethal of the natural biological neurotoxins known. LD of botulinum toxin (refined neurotoxin complex) serotype A in mice₅₀About 50 picograms. A single unit of botulinum toxinSite (U) is defined as LD after intraperitoneal injection in female Swiss Webster mice weighing 18-20 grams each₅₀. Seven immunologically distinct botulinum neurotoxins have been characterized, each of these toxins being botulinum neurotoxin serotype A, B, C₁D, E, F and G, which are distinguished by neutralization with serotype-specific antibodies. The different serotypes of botulinum toxin vary with the animal species they affect and the severity and duration of the paralysis they induce. For example, when the rate of paralysis produced in rats is determined, BoNt/A has been determined to be 500 times more potent than botulinum toxin serotype B (BoNT/B). In addition, it has been determined that BoNt/B is not toxic to primates at a dose of 480U/kg, which is 12 times the LD of BoNt/A for primates₅₀The value is obtained. Botulinum toxin, which binds significantly with high affinity to cholinergic motor neurons, is transported into the neuron and blocks the release of acetylcholine.

Botulinum toxin has been used clinically 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. It is clear that non-serotype A botulinum toxin serotypes are less potent and/or have a shorter duration of activity compared to BoNt/A. The intramuscular clinical effects of botulinum toxin, such as BoNt/A, are noted within a few hours. Thus, as determined, upon intramuscular injection, it is important to note that most, if not all, of the botulinum toxins produce significant muscle paralysis within one day after injection (as determined by the mouse Digital Abduction Score (DAS)). Aoki k.r., preclinic Update on BOTOX (botulinum toxin type a) -refined neurotoxin complex relative to other botulinum toxin preparations Eur j. neuron 1999, 6(suppl 4): S3-S10. The duration of remission obtained by a single intramuscular injection of BoNt/a is generally on average about 3 months. Botulinum toxins, including botulinum toxin type A, having a shortened in vivo period of biological activity are set forth in co-pending U.S. patent application serial No. 09/620840, which is hereby incorporated by reference in its entirety.

While all botulinum toxin serotypes significantly inhibit the release of the neurotransmitter acetylcholine at the neuromuscular junction, they act by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum serotypes A and E both cleave a 25 kilodalton (kD) synaptosome associated protein (SNAP-25), but they target different amino acid sequences within this protein. BoNT/B, D, F and G act on vesicle-associated proteins (VAMP, also known as synaptophysin), where each serotype cleaves the protein at a different site. Finally, botulinum toxin serotype C has been shown to₁(BoNT/C₁) Capable of cleaving syntaxin and SNAP-25. These differences in mechanism of action can affect the relative intensity and/or duration of action of the various botulinum toxin serotypes.

Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and involves at least three steps or stages. In the first step of the process, the toxin binds with high affinity to the presynaptic membrane of the target neuron through specific interactions between the H chain and cell surface receptors; this receptor is believed to be different for each serotype of botulinum toxin and tetanus toxin. The carboxy-terminal fragment of the H chain, Hc, appears to be important for targeting the toxin to the cell surface.

In the second step, the toxin passes through the serosal membranes of the poisoned cells. The toxin is first engulfed by the cell by receptor-mediated endocytosis and endosomes are formed which contain the toxin. The toxin then escapes from the endosome into the cytoplasm of the cell. The last step is considered to be represented by the amino-terminal fragment of the H chain-H_NMediated, upon exposure to a pH of about 5.5 or less, it can trigger a morphological change in the toxin. Endosomes are known to have a proton pump that lowers the pH in the endosome. This morphological change exposes the hydrophobic nature of the toxinResidues which allow the toxin to embed itself into the membrane of the endosome. The toxin is then transported through the membrane of the endosome into the cytoplasm.

The last step in the mechanism of botulinum toxin activity appears to involve the critical intracellular exocytosis protein cleavage by the L chain. All toxic activities of botulinum and tetanus toxins are contained in the L chain of the holotoxin; the L chain is a zinc (Zn + +) endopeptidase that selectively cleaves proteins necessary for recognition and enables vesicles containing neurotransmitters to dock with the cytoplasmic surface of the plasma membrane, and to fuse with the plasma membrane. Tetanus neurotoxin, botulinum toxin/B,/D,/F, and/G can cause degradation of synaptobrevin (also known as vesicle-associated membrane protein (VAMP)), a synaptobrevin membrane protein. Most of the VAMP present on the cytoplasmic surface of the synaptic vesicle as a result of any one of these cleavage results can be removed. Each toxin can specifically cleave a different bond.

For all seven known botulinum toxin serotypes, the molecular weight of the botulinum toxin protein molecule is approximately 150 kD. Interestingly, the botulinum toxin is released by Clostridium, which is a complex form comprising the 150kD botulinum toxin protein molecule in combination with non-toxin proteins. Thus, the BoNt/A complex can be produced by Clostridium, such as in the form of 900kD, 500kD and 300 kD. BoNT/B and C₁It appears to be produced only as a 500kD complex. BoNT/D can be produced as a 300kD and 500kD complex. Finally, BoNT/E and F can only be produced as about 300kD complexes. Finally, BoNT/E and F can only be produced as about 300kD complexes. The complex (i.e., having a molecular weight greater than about 150kD) is believed to comprise one non-toxin haemagglutinin protein and one non-toxin non-haemagglutinin protein. The two non-toxin proteins (which together with the botulinum toxin molecule form the relevant neurotoxin complex) can provide stability against the denaturing effects of the botulinum toxin molecule and when the toxin is ingestedCan be administered without the influence of digestive tract acid. In addition, it is possible that the larger (molecular weight greater than about 150kD) botulinum toxin complex may cause the botulinum toxin to exit the intramuscular injection site of the botulinum toxin complex at a slower diffusion rate.

Botulinum toxin has been shown to inhibit potassium cation induced release of acetylcholine and norepinephrine from brainstem tissue primary cell cultures in vitro studies. In addition, botulinum toxin has been reported to inhibit the release of glycine and glutamate induced in the main culture of spinal cord neurons, and there have also been reports of botulinum toxin inhibiting the release of various neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate within brain synaptosome preparations.

BoNt/a is obtained by implanting clostridium botulinum in a fermenter and growing a culture thereof, then capturing it and purifying the fermented mixture by known methods. All botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or cleaved by proteases to become neuroactive materials. Bacterial strains capable of producing botulinum toxin serotypes A and G have endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in a form that has significant activity. In contrast, botulinum toxin serotype C₁D and E are synthesized by using strains which are not proteolytically active and therefore are generally inactive when recovered from culture. Serotypes B and F are synthesized using both proteolytic and nonproteolytic strains and can therefore be recovered in active form or can be recovered in inactive form. However, even strains that produce proteolysis, such as the BoNt/B serotype, cleave only a portion of the toxin produced. The exact ratio of cleaved to uncleaved molecules depends on the time of incubation and the temperature of incubation. Thus, a certain percentage of any one agent, such as the BoNt/B toxin, may be inactive, which may be known as BoNt/B was much less effective than BoNt/A. The presence of inactive botulinum toxin molecules in a clinical preparation contributes to the total protein load of the preparation, which is associated with an increase in antigenicity, but is not helpful for its clinical efficacy. Furthermore, it is known that upon intramuscular injection, BoNt/B has a shorter duration of active action and is also less potent than BoNt/a at the same dose level.

BoNt/A has been reported to have been used in the clinic for the following applications:

(1) about 75-125 units of BOTOX * per intramuscular injection (of various muscles)¹For use in the treatment of cervical dystonia; (¹Available from Allergan, Inc., of Irvine, California, registered trademark BOTOX *)

(2) BOTOX * with 5-10 units per intramuscular injection for treating glabellar lines (sulcus of the eyebrow) (5 units per intramuscular injection for intramusculus glabellar and 10 units per intraeyebrow muscle);

(3) treating constipation by intramuscular injection of about 30-80 units of BOTOX * into the puborectal muscle;

(4) blepharospasm is treated by intramuscular injection of about 1-5 units of BOTOX * per injection to the lateral palpebral anterior muscle of the upper and lower eyelids.

(5) For treatment of strabismus, the extraocular muscles are intramuscularly administered with about 1-5 units of BOTOX *, the amount of injection being variable depending on the size of the injected muscle and the degree of muscle paralysis desired (i.e., the amount of diopter correction number desired).

(6) To treat upper limb spasms following stroke, BOTOX * may be intramuscularly in five different upper limb flexors as follows:

(a) deep flexor digitorum profundus: 7.5U to 30U

(b) Superficial flexor digitorum sublimus (flexo rdigithorum): 7.5U to 30U

(c) Carpal flexor carpi ulnaris (flexor carpi ulnaris): 10U to 40U

(d) Wrist flexor carpi radialis: 15U to 60U

(e) Biceps brachii muscle: 50U to 200U. Each of the five muscles listed were injected during the same treatment period so that the patient could receive 90U to 360U of BOTOX * by intramuscular injection during each treatment period for the upper limb flexors.

The successful treatment of various clinical conditions by BoNt/a has led to interest in other botulinum toxin serotypes. Two commercially available BoNT/a preparations (BOTOX * and Dysport *) and BoNT/B and F preparations (both from Wako Chemicals, japan) have been studied in order to determine pre-clinical local muscle weakness efficacy, safety and potential antigenicity. Botulinum toxin formulations were injected into the top of the right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscle weakness was assessed using a mouse toe abduction score test (DAS). Calculation of ED from dose response curves₅₀The value is obtained. In addition, some mice were used to determine LD by intramuscular injection₅₀And (4) dosage. Through LD₅₀/ED₅₀To calculate the therapeutic index. Groups of mice were injected on hind limbs with BOTOX * (5.0 to 10.0 units/kg) or BoNt/B (50.0 to 400.0 units/kg) and then tested for muscle weakness and increased water consumption, a well-established model for dry mouth. Potential antigenicity was assessed by a monthly intramuscular test with rabbits (BoNt/B of 2.0 or 8.7 units/kg or BOTOX * of 3.0 units/kg). The maximum degree and duration of muscle weakness was associated with all serotype doses. DASED₅₀The values (units/kg) are as follows: BOTOX *: 6.7, Dysport *: 24.7, BoNt/B: 11.8 to 244.0, BoNT/F: 4.3. BOTOX * has a longer duration of action than BoNt/B or BoNt/F. The therapeutic index values are as follows: BOTOX *: 10.5, Dysport *: 6.3, BoNt/B: 4.8. although BoNt/B was less effective in sparing muscle, water consumption was higher in mice injected with BoNt/B than in mice injected with BOTOX *. Four months after injection, two of four rabbits (treated with 1.5 ng/kg) and four rabbitsFour (treated with 6.5 ng/kg) antibodies were raised against BoNt/B. In a separate study, none of the 9 rabbits treated with BOTOX * produced antibodies to BoNt/A. DAS results indicate that BoNt/A has a relative peak intensity equal to BoNt/F, which is stronger than BoNt/B. As far as the duration of action is concerned, BoNt/A is longer than BoNt/B, and BoNt/B is longer than BoNt/F. As indicated by the therapeutic index values, the two commercial formulations of BoNt/A (BOTOX * and Dysport *) were different. The observed increase in water consumption behavior 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 the doses of the other serotypes tested must be increased in order to obtain efficacy comparable to that of BoNt/a. Increasing the dosage may involve safety issues. Furthermore, serotype B was more antigenic than BOTOX * in rabbits, probably because of the higher protein load injected to achieve an effective dose of BoNt/B.

Tetanus neurotoxin acts primarily in the central nervous system, whereas botulinum neurotoxin acts at the neuromuscular junction; both may act by inhibiting the release of acetylcholine from the axons of affected neurons into the synapses, leading to paralysis. The toxic effects of the affected neurons are long-lasting and, to date, are considered irreversible. Tetanus neurotoxin is known to exist as an immunologically distinct serotype.

Acetylcholine

It is generally only a single type of small molecule neurotransmitter that is released by various types of neurons within the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many regions of the brain, but in particular 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 (sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers of the sweat glands, piloerection (primoerector) muscles and some blood vessels are cholinergic, and most of the postganglionic neurons of the sympathetic nervous system release the neurotransmitter norepinephrine. In most cases, acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects on certain peripheral parasympathetic nerve endings, such as inhibiting the heart through the vagus nerve.

The output signals of the autonomic nervous system are transmitted into 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 medial and lateral corners of the spinal cord. The preganglionic sympathetic nerve fibers extending from the cell body, synapses with postganglionic neurons are located in the sympathetic ganglia of the paraspinal (paravertebral) or in the ganglia in front of the vertebrae (prevtebral). Because the preganglionic neurons of both the sympathetic and parasympathetic nervous systems are cholinergic, the use of acetylcholine for this ganglion simultaneously excites the postganglionic neurons of both the sympathetic and parasympathetic nerves.

Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. Muscarinic receptors are found in all effector cells stimulated by postganglionic neurons of the parasympathetic nervous system as well as those stimulated by postganglionic cholinergic neurons of the sympathetic nervous system. Nicotinic receptors are found in the synapses between the preganglionic and postganglionic neurons of both sympathetic and parasympathetic nerves. The nicotinic receptor is also present on 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. Many non-neuronal secretory cells, such as the adrenal medulla (and the PC12 cell line) and pancreatic islet cells, can release catecholamines and insulin, respectively, from a large number of dense nuclear vesicles. The PC12 cell line is a pure line of rat pheochromocytoma cells that is widely used as a tissue culture model for the development of the sympathoadrenal gland. In vitro, the toxin inhibits the release of both compounds from both types of cells, either by osmosis (e.g., by electroporation) or by direct injection of botulinum toxin into denervated cells. Botulinum toxin is also known to block the release of the neurotransmitter glutamate from cortical synaptosomal cell cultures.

Neuromuscular junctions are formed in skeletal muscle by the proximity of axons to muscle cells. Signals transmitted through the nervous system produce action potentials at the terminal axon, which activate ion channels and lead to the release of the neurotransmitter acetylcholine from the intraneural synaptic vesicle, for example at the motor endplate at the neuromuscular junction. The acetylcholine binds to acetylcholine receptor proteins on the surface of the muscle end plates through the extracellular space. Once sufficient binding has occurred, the action potential of the muscle cell can cause specific membrane ion channels to change, resulting in muscle cell contraction. Acetylcholine is then released from the muscle cells and metabolized by cholinesterase in the extracellular space. The metabolite is recycled back into the terminal axon to be reprocessed into additional acetylcholine.

As discussed above, current methods of treating damaged muscles are still inadequate. There is a need for an improved method of treating damaged muscle.

Summary of The Invention

According to the present invention, an effective method for treating an injured muscle comprises the step of locally administering a therapeutically effective amount of a neurotoxin into or around the injured muscle in vivo. The neurotoxin provides a temporary chemodenervation effect to the injured muscle and reduces the contraction of the muscle. The purpose of the present invention is to promote healing of damaged muscles and to quickly restore their function. The damaged muscle may be, for example, a sprained muscle. In one embodiment, the neurotoxin is administered intramuscularly or subcutaneously. In another embodiment, the step of administering the neurotoxin is performed before and/or after physical therapy and/or surgery.

Also in accordance with the present invention, the step of administering the neurotoxin is performed immediately after the muscle is injured, or immediately thereafter as is practicable. In one embodiment, the neurotoxin can effectively immobilize or substantially immobilize the damaged muscle at least during phase 1 and/or phase 2 of the damaged muscle repair process.

According to the present invention, the neurotoxin can comprise a targeting component, a therapeutic component and a transport component. The targeting component may be associated with a presynaptic motor neuron. In one embodiment, the targeting component can comprise a butryric toxin, a tetanus toxin, or A, B, C₁The carboxy-terminal fragment of the heavy chain of botulinum toxin type D, E, F, G or a variant thereof. The therapeutic component may interfere with or modulate neurotransmitter release from the neuron or its processing. In one embodiment, the therapeutic component comprises a butryotoxin, tetanus toxin, or A, B, C₁And a light chain of botulinum toxin type D, E, F, G or a variant thereof. The transport component can facilitate transport of at least a portion of the neurotoxin, such as a therapeutic component, into the cytoplasm of a target cell. In one embodiment, the transport component can comprise a butynotoxin, a tetanus toxin, or A, B, C₁An amino-terminal fragment of the heavy chain of botulinum toxin type D, E, F, G or a variant thereof.

Also according to the invention, the neurotoxin is a botulinum toxin type A, B, E and/or F. In a preferred embodiment, the neurotoxin used to treat damaged muscle is botulinum toxin type A. Indeed, botulinum toxin type A is preferred because of its commercial availability, its known clinical use, and the successful use in the treatment of muscle damage according to the present invention, as disclosed herein. The use of between about 0.1U/kg and about 3OU/kg of botulinum toxin type A and between about 1U/kg and about 150U/kg of botulinum toxin type B is within the scope of the actual process disclosed herein. With respect to other botulinum toxin serotypes (including type E and type F toxins), as described herein, the U/kg dose used ranges from about 0.1U/kg to about 150U/kg.

Also according to the invention, the neurotoxin can be produced recombinantly.

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

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

Definition of

The following definitions are provided and applied herein.

"about" means about or near, and references herein to a stated value or range means ± 10% of the stated or claimed value or range.

"heavy chain" refers to the heavy chain of a clostridial neurotoxin. It preferably has a molecular weight of about 100kDa and is referred to herein as the H chain or H.

“H_N"refers to a fragment of the H chain from a clostridial neurotoxin that corresponds approximately to the amino terminal fragment of the H chain (preferably having a molecular weight of about 50kDa), or a portion of that fragment that corresponds to the entire H chain. It is considered to be included in the traversing fineA portion of a native or wild-type clostridial neurotoxin involved in the transport of the L chain of the intracellular endosomal membrane.

"Hc" refers to a fragment of the H chain from a clostridial neurotoxin that is approximately equivalent to the carboxy-terminal fragment of the H chain (about 50kDa), or a portion corresponding to that fragment in the intact H chain. It is believed to be immunogenic and comprises a portion of the native or wild-type clostridial neurotoxin involved in presynaptic binding with motor neurons with high affinity.

"damaged muscle" includes sprains, tears or tears, as well as muscles with bruises (bruises), lacerations, ischemia or rupture.

"light chain" refers to the light chain of clostridial neurotoxin. It preferably has a molecular weight of about 50kDa and is referred to as the L chain, L or proteolytic region (amino acid sequence) of the clostridial neurotoxin. The light chain is believed to be a potent inhibitor of neurotransmitter release when released into the cytosol of the target cell.

By "topical administration" is meant the direct administration of a drug to a site on or near a site on or in the body of an animal where it is desired that the drug exert some biological effect. Topical administration excludes administration by the systemic route, such as intravenous or oral administration.

"neurotoxin" refers to a chemical entity that interferes with or modulates at least one function of a neuron. A "neurotoxin" can be naturally occurring or can be artificial. In addition, the "neurotoxin" can be a small molecule, a large molecule, a polypeptide, a conjugated polypeptide or a mixture thereof.

"variant" refers to a chemical entity that differs slightly from the parent chemical entity but still has a biological effect. The biological effect of the variant may be substantially the same as or better than that of the parent. For example, a variant light chain of botulinum toxin having at least one amino acid substituted, modified, deleted or added can have the same or greater ability to prevent neurotransmitter vesicle release. In addition, the biological effects of the variant may also be reduced. For example, a variant light chain of botulinum toxin type A with the leucine motif removed can have a shorter biological persistence than the parent (or native) botulinum toxin type A light chain.

Description of the invention

In a primary embodiment, an effective method of the present invention for treating damaged muscle can comprise the step of topically administering a therapeutically effective amount of a neurotoxin to the damaged muscle. Preferably, the damaged muscle is a strained muscle.

Sprained damage to skeletal muscle can be classified as shear injury. In a shear injury, not only the muscle fibers are torn, but also the mysial sheath. The process of muscle repair begins almost immediately after muscle injury. The repair process of shear injury can be divided into three stages.

Stage 1 is the destruction stage, which is characterized by the formation of hematomas, myofiber necrosis, and inflammatory cell responses. After a sprain, rupture sites of other healthy muscles often occur near their distal tendon junctions (MTJs). The ruptured muscle fibers contract and form a gap between the remnants. Since the blood vessels of skeletal muscle are abundant, bleeding from the torn blood vessels is inevitable, and the gap is filled with hematoma and then replaced with scar tissue. In a shear injury, the mechanical force tears the entire muscle fiber, damaging the serosa of the muscle fiber and leaving a sarcoplasmic opening at the end of the remnant. Because the muscle fibers are long, stringy cells, the necrosis extends from such a site throughout the length of the ruptured muscle fiber. Blood vessels are also torn in shear injury; thus, inflammatory cells carried by the blood immediately enter the site of injury and induce inflammation. After injury, phase 1 will last for about 2 to 3 days.

The 2 nd stage is the repair stage, which consists of phagocytic lysis of necrotic tissue, regeneration of muscle fibers, generation of connective tissue scars, and ingrowth of capillaries. A key step in the regeneration of damaged muscle tissue is the vascularization of the damaged area. Repair of vascular recruitment is essential for the regeneration of damaged muscle. New capillaries extend from the surviving large vessels and grow toward the center of the lesion. These new capillaries help provide an adequate supply of oxygen to the regeneration zone.

The 3 rd stage is a remodelling stage consisting of maturation of the regenerated muscle fibres, contraction and organization of scar tissue, and functional recovery of the repaired muscle. After phase 1, phase 2 (repair) and phase 3 (retrofit) often occur simultaneously and last from about 2 days to about 6 weeks.

In one embodiment of the invention, the neurotoxin is administered topically to immobilize damaged muscles to promote healing, preferably by intramuscular administration. The topical administration of the disclosed neurotoxin also reduces pain due to muscle damage. Preferably, the neurotoxin is administered at or immediately after the injury. In a preferred embodiment, the neurotoxin is effective to immobilize a damaged muscle during the destruction phase (phase 1) to prevent re-rupture of the muscle.

Without wishing to be bound by any particular theory of mechanism of action, it is believed that immobilization during the repair and/or remodelling phase is beneficial because it may allow faster and stronger ingrowth of capillaries in the damaged area and may allow better regeneration and orientation of muscle fibres. Thus, in one embodiment, there is no immobilization of the neurotoxin during the repair phase (phase 2) and/or the remodeling phase (phase 3). In a more preferred embodiment, the neurotoxin is administered during phase 1, which is effective to immobilize damaged muscle, but the neurotoxin is not administered during phases 2 and 3 of the repair process. For example, if the neurotoxin is injected immediately after injury, preferably intramuscularly, the neurotoxin immobilizes the injured muscle, preferably within a period of about 3 days after administration. Or the neurotoxin can have an immobilization effect only to the extent that the patient experiences little or no pain during the underlying activity after application to the damaged muscle. When such a critical point is reached, the patient should be encouraged to initiate a positive and gradual activity.

In another embodiment of the invention, the neurotoxin is effective to immobilize damaged muscle during all of the 1 st to 3 rd stages and during the subsequent recovery period of muscle damage.

Neurotoxins, such as certain botulinum toxins, which require less than about one to about seven days to exhibit significant clinical muscle paralysis and/or in which the muscle paralysis can persist for a period of several months after injection, are within the scope of the present invention, and can be used to treat relatively severe or prolonged muscle damage or situations requiring long-term muscle immobilization for healing purposes.

In a main embodiment, the neurotoxin is a neuromuscular blocking agent. Table 1 lists, without limitation, some neuromuscular blocking agents and their possible sites of action. In one embodiment, a neuromuscular blocking agent having the ability to immobilize a muscle, preferably an injured muscle, for at least about 5 days, preferably at least about 3 days, is administered to treat an injured muscle. In a preferred embodiment of the invention, the neurotoxin is a botulinum toxin due to the known use and clinical safety of botulinum toxin, such as botulinum toxin E, for example, for the treatment of muscle disorders such as muscle spasms. In a particularly preferred embodiment of the present invention, particularly for severe, or third degree muscle damage, the botulinum toxin administered topically is a botulinum toxin type E. Botulinum toxin type A may also be used in these embodiments.

TABLE 1

Compound (I)	Site of action relative to NMJ	Pharmacological classes
			Aconitine Adenoegulin (available from FrogPhotodeuusaBicolor) adenosine agonist adenosine antagonist adenosine modulator Adenoergic drug Toxoid-A antiepileption antianxiety drug Atacuum Atacutum besylate (Carcinon) baclofen (Lioresal. RTM., Geigy; Intathecal, medicinal Neurology; generic, Athenna, Biocraft, Warner Chilcott)	Presynaptic&Postsynaptic presynaptic&Postsynaptic presynaptic&Postsynaptic presynaptic CNS presynaptic&postCNS postsynaptic presynaptic	Antisense technology of ACh esterase inducer sodium channel activator adenosine receptor modulator adenosine alpha adrenergic neuro drug AchR antagonist antiepileptic agent important messenger in neurotransmitter release, receptor production or specific protein

Bacteria, plant and fungus product curdline benzyl piperidine plant neurotoxin cygarterToxin-beta (beta-BuTX) bupivacaine captopril (Capoten.RTM., Squibb; Capzide.RTM., Squibb) acetylcholine+Transferase inhibitor cholinesterase inhibitor dinoflagellate toxin conotoxin MI (alpha conotoxin) conotoxin-mu.GIIIA (mu-CT) conotoxin-omega.GVIA (omega-CT) curare dantrolene sodium (dantrolene, P-D)&G) Desmodium bromide-decadimonium dendroaspin

Presynaptic and postsynaptic gaps in presynaptic and postsynaptic gaps and presynaptic and postsynaptic gaps in presynaptic and postsynaptic gaps

Sodium channel activators Ach esterase inhibitors (unconventional) a variety of PLA2 and voltage sensitive potassium channel blockers. Snake venom local anesthetic, Myotoxin, from Agkistrodon Halys, anti-hypertensive ACE inhibitor Zinc endopeptidase inhibitor CAT inhibitor Ach esterase inhibitor sodium channel AchR antagonist Na+Ca channel blockers in neurons only+Channel blocker AChR antagonist non-depolarizing skeletal muscle relaxant AChR antagonist ganglion blocker potassium channel blocker

Diaminopyridine (3-DPA) diaza-doxa-chloride (Nuroma. RTM., Burroughs Wellcome) Adriamycin (Hydroxydaunorubicin, Adria; Rubex, Immunex; CetusOncology) Epibat idine dihydrochloride non-urethane (febantel, Carter-Wallace logic to Schering-Plough) Methanomycin gabapentin (Neurotontin, Parke-Davis) Coiaminoammonium quinotoxin hexahydroaza * acetamide and other classes of chemical substance huperzine A insect venom ion channel blocker Latrotoxin-alpha

Pre-synaptic and post-synaptic pre-synaptic postsynaptic CNS postsynaptic pre-synaptic cleft pre-and post-synaptic pre and post-synaptic cleft pre

Reversible botulinum toxin intoxication anxiolytic AchR antagonist non-depolarizing muscle relaxant Myotoxin (Myotoxin) chemomyoablation AchR agonist antiepileptic angiotensin I convertase inhibitor antiepileptic GABA analogue AchR antagonist sodium channel activator Ach releasing agent Ach esterase inhibitor channel blocker channel agonist calcium ionophore

Lidocaine, procaine, mepivacaine, etc. (dunp 996, dupont merck) sarcandra toxin and the analogue marine natural product guaiacol glyceryl carbamate ether (Shujingling, RobinsCo.) medobulin micaroline chloride mivaklone (mivacro. rtm., BWBW1090U, burroughs wellcom) anti-NMJ component modified clostridiatoxin monoclonal antibody muscarinic agonists and antagonists neosaxitoxin neuromagnatoxin neuromuscular blockers

Pre-presynaptic post-presynaptic postsynaptic pre-and post-presynaptic postsynaptic

Irreversible AchR antagonist CNS inhibitors of local anaesthetic Ach release enhancers, non-depolarizing muscle relaxant AchR antagonist muscle relaxant Ach release inhibitor receptors, agroins, neurotransmitters, plasmaMembrane fraction, inactivated enzyme, and the like, muscarinic CNS agonist antagonist sodium channel blockers autonomic ganglionic AchR blockers (NMJ null) AChR antagonist AChR depolarizers

Neurotoxin bromopankangnin (desogestrel) pankangnin-3-OH metabolite (desogestrel) papaverine hydrochloride (30mg/ml) physostigmine and analogs of Pipercuronium (Arduan, desogestrel) presynaptic nerve terminal receptor short-term neurotoxin alpha beta-cycloprotoxin (beta-BuTX) succinylcholine chloride (succinylcholine chloride, BurroughhsWecomole) tetanus toxin transporter Tetrahydroaminoacridine (THA) tetrodotoxin (tetrodoxin) thiogatran (Novo disk) transglutaminase inhibitor or induction of prevention of diazepam

Pre-and post-synaptic cleft postsynaptic presynaptic postsynaptic cleft presynaptic postsynaptic presynaptic and postsynaptic CNS pre-and postsynaptic clefts

A variety of AChR antagonist non-depolarizing muscle relaxants AChR antagonists non-depolarizing muscle relaxants smooth muscle relaxants AchR esterase inhibitors AChR antagonists non-depolarizing muscle relaxants any extraneuronal or intraneuronal receptor AChR antagonists at the nerve terminals the anti-epileptic GABA uptake inhibitor, the enzyme diazepam, the AChR inhibitor, the EAA release inhibitor, the skeletal muscle relaxant, the sodium channel blocker, the AChR inhibitor, the snake venom AChR receptor agonist depolarizing

Zinc endopeptidase and other proteases delivered by botulinum toxin or tetanus toxin transporters from vancomycin (vecuronium bromide, desogestrel) vancomycin-3-OH metabolite (desogestrel) Veratridine vigabatrin (Sabril, MarionMerrell Dow) Vesamicol and other drugs with the same mechanism

Postsynaptic presynaptic CNS presynaptic

AChR antagonist non-depolarizing muscle relaxant sodium channel activator antiepileptic drugs (irreversible) GABA metabolic inhibitor Ach vesicle transport inhibitor enzyme reduces neurotransmitter release

In a primary embodiment, the neurotoxin can comprise a targeting component, a therapeutic component, and a transport component. The targeting component can bind to a presynaptic motor neuron. In one embodiment, the targeting component may comprise a butryotoxin, tetanus toxin, A, B, C₁The carboxy-terminal fragment of the heavy chain of botulinum toxin type D, E, F, G or a variant thereof. In a preferred embodiment, the targeting component may comprise a carboxy terminal fragment of botulinum toxin type A.

The therapeutic component may directly interfere with or modulate neurotransmitter release from the cell or its processing. In one embodiment, the treatmentThe components comprise butyribacterium toxin, tetanus toxin and A, B, C₁And a light chain of botulinum toxin type D, E, F, G or a variant thereof. In a preferred embodiment, the therapeutic component may comprise a light chain of botulinum toxin type having a short biological duration, such as less than about 5 days, preferably less than about 3 days. Preferably, such light chains may be those of botulinum toxin types E or F. Alternatively, the light chain may be that of botulinum toxin type A.

The transport component can facilitate transfer of at least a portion of the neurotoxin, such as a therapeutic component, into the cytoplasm of a target cell. In one embodiment, the transport component comprises a butynotoxin, tetanus toxin, A, B, C₁An amino-terminal fragment of the heavy chain of botulinum toxin type D, E, F, G or a variant thereof. In a preferred embodiment, the transport component comprises an amino-terminal fragment of the heavy chain of botulinum toxin type A.

In one embodiment, the targeting component comprises a carboxy terminal fragment of the heavy chain of botulinum toxin type E or F, the therapeutic component comprises the light chain of botulinum toxin type E or F and the transport component comprises an amine terminal fragment of the heavy chain of botulinum toxin type E or F. In a preferred embodiment, the neurotoxin comprises botulinum toxin type E. In another preferred embodiment, the neurotoxin comprises botulinum toxin type F. In yet another embodiment, the neurotoxin comprises a mixture of botulinum toxins types E and F.

In one embodiment, the targeting component comprises a carboxy terminal fragment of the heavy chain of botulinum toxin type a, the therapeutic component comprises the light chain of botulinum toxin type a and the transport component comprises an amine terminal fragment of the heavy chain of botulinum toxin type a. In a preferred embodiment, the neurotoxin of the present invention comprises botulinum toxin type A. One suitable botulinum toxin type A for use herein is BOTOX * (Allergan, Inc., Irvine, California.)

Although in one embodiment, the neurotoxin of the present invention is treated by immobilizing a damaged muscle, the neurotoxin can be administered to a damaged muscle to reduce pain and/or spasticity. In another embodiment, the neurotoxin is capable of immobilizing an injured muscle and of reducing pain associated with the injured muscle. In a preferred embodiment, a neurotoxin, such as a botulinum toxin type E, or more preferably a botulinum toxin type A, is applied to a strained muscle to immobilize the muscle and/or reduce pain associated with the muscle.

Of course, the ordinary professional medical provider may determine the appropriate dosage and frequency of administration to achieve the optimal clinical effect. That is, one of ordinary skill in the medical arts can administer an appropriate amount of neuromuscular blocking agent at an appropriate time to facilitate effective fixation of damaged muscles. The dose of neurotoxin administered depends on various factors including the size of the muscle, the severity of the muscle injury. In a preferred embodiment, the dose of neurotoxin used can immobilize the damaged muscle for a period of time that does not exceed phase 1 of the repair process. In various methods of the present invention, about 0.1U/kg to about 15U/kg of a botulinum toxin type A can be administered to the injured muscle. Preferably, about 1U/kg to about 20U/kg of a botulinum toxin type A can be administered to the injured muscle. The use of between about 0.1U/kg and about 30U/kg of botulinum toxin type A and between about 1U/kg and about 150U/kg of botulinum toxin type B is also within the scope of the disclosed embodiments. For other botulinum toxin serotypes, including E and F toxins, dosages in the range of about 0.1U/kg to about 150U/kg can be used, as described herein.

Although intramuscular injection is the preferred route of administration, other routes of topical administration, such as subcutaneous administration, may also be used.

In another main embodiment, the method for treating damaged muscle according to the present invention further comprises other steps as described below. These additional steps may be performed prior to, with or after administration of the neurotoxin, preferably administration of the neurotoxin to the damaged muscle. For example, the proposed treatment for a muscle sprain includes resting, icing, pressing and lifting. These four steps (or processes) have the same purpose. They minimize bleeding from the ruptured blood vessel to the site of the rupture. Doing so will prevent the formation of a wide range of hematomas, which will directly affect the size of the scar tissue at the end of the regeneration event. Small hematomas and accumulation of edema in the fracture site and limited interstitial spaces can also shorten the ischemic period in granulation tissue, which in turn can promote regenerative effects.

Other additional steps may also be used in the treatment of damaged muscles. In one embodiment, the additional step includes the use of non-steroidal anti-inflammatory drugs (NSAIDs), ultrasound therapy, hyperbaric oxygen, and in severe injury, surgery may also be performed. NSAIDs should be part of early treatment and should be used immediately after injury. Short term use of NSAIDs in the early stages of healing can reduce inflammatory cell responses and have no adverse effect on the elongation and contractility of damaged muscles.

In another embodiment, the additional step comprises the use of ultrasound therapy. Ultrasound therapy is widely recommended and used for the treatment of muscle sprains. Ultrasound treatment is believed to promote muscle regeneration during the proliferative phase.

In another embodiment, the additional step includes the use of high pressure oxygen. Hyperbaric treatment of rabbits in the early stages of repair is known to significantly improve the end result. It is believed that such hyperbaric treatments are helpful in other mammals, such as humans, for example to accelerate muscle regeneration.

In another embodiment, the additional step comprises surgical intervention. Surgical treatment of muscle injuries should be used for the most severe injuries, since in most cases conservative treatment can be used with good results. The surgical treatment is only recommended for the following cases: (1) large intramuscular hematomas, (2) third degree sprains or tears with little or no muscle discomfort, and (3) second degree sprains if more than half of the muscle belly is torn.

In another main aspect of the invention, the components of at least one neurotoxin can be prepared using recombinant techniques. The technique includes the steps of obtaining genetic material from a pure line of DNA obtained from natural material or obtaining a synthetic oligonucleotide sequence that can encode one of the components, e.g., a therapeutic, transport and/or targeting component. The genetic construct may first be amplified by fusion of the genetic construct with a cloning vector, such as a phage or plasmid, and incorporated into a host cell. The cloning vector is then seeded into a host, preferably E.coli (E.coli's). After expression of the recombinant gene in a host cell, the resulting protein is isolated using conventional techniques. The expressed protein may comprise all three components of the neurotoxin. For example, the expressed protein may comprise the light chain of botulinum toxin type E (therapeutic component), the heavy chain of botulinum toxin type B, preferably H_N(transport component), and the Hc of botulinum toxin type a, which can selectively bind to motor neurons. In one embodiment, the expressed protein may comprise a portion of all three components of the neurotoxin. In this case, the components may be chemically linked using techniques well known in the art.

Recombinant production of these neurotoxins has many advantages. For example, the preparation of neurotoxins from anaerobic clostridium cultures is a cumbersome and time-consuming process involving a multi-step purification scheme involving several protein precipitation steps, and prolonged or repeated crystallization of the toxin or column chromatography in several steps. Notably, the high toxicity of the product necessitates that the process be completed under strictly sealed conditions (BL-3). During fermentation, the folded single chain neurotoxin is activated by endogenous clostridial proteases through a process called nicking. This involves the removal of approximately 10 amino acid residues from a single chain to form a double-stranded form in which the two chains are still covalently linked by an intrachain disulfide bond.

The notched neurotoxin is more active than the non-notched form. The amount and precise location of the incision will vary with the serotype of the bacterium from which the toxin is produced. Thus, differences in single chain neurotoxin activation and differences in the yield of nicked toxin are caused by differences in the type and amount of proteolytic activity produced by a given strain. For example, greater than 99% of Clostridium botulinum single-chain neurotoxins type A are activated by Hall A strains of Clostridium botulinum, while type B and E strains produce toxins with lower activation amounts (0 to 75%, depending on fermentation time). Thus, the highly toxic mature neurotoxin is a major part of the commercially produced neurotoxins as therapeutic neurotoxins.

Therefore, the degree of activation of the engineered clostridial toxins is an important consideration in the production of these substances. It would be a major advantage if neurotoxins such as botulinum toxin and tetanus toxin could be recombinantly expressed in rapidly growing bacteria (e.g., xenogeneic E.coli cells) in high yields in relatively non-toxic single-stranded (or single-stranded with reduced toxicity) forms that are safe, easily isolated and readily converted to fully active forms.

Since safety is the most fundamental consideration, previous work has focused on the expression and purification of the respective H and L chains of tetanus toxin and botulinum toxin in E.coli; these isolated chains are non-toxic in nature; see Li et al, Biochemistry 33: 7014 — 7020 (1994); zhou et al, Biochemistry 34: 15175-15181(1995), which is incorporated herein by reference. After the peptide chains are separately produced and under tightly controlled conditions, the H and L subunits can be joined by oxidized disulfide bonds to form neuroparalytic duplexes.

The following non-limiting examples provide preferred methods for treating damaged muscle and for producing recombinant neurotoxins, preferably botulinum toxins. The process for making recombinant botulinum toxin is described in examples 4-8 below and is the same as that described in Dolly et al, International patent application WO 95/32738, the disclosure of which is incorporated herein by reference in its entirety.

Example 1

Treatment of ruptured biceps tendon

Rupture of the biceps brachii occurs generally at the proximal end of the biceps and involves the distal end (longhead) of the biceps. The muscle may break at the distal attachment on the radius, but this is rare. Most commonly, rupture occurs in adults over 40 years of age with a long history of shoulder pain following the collision syndrome. For a long time, the tendons have been subject to wear and become very fragile and eventually break partially or totally. In any event, the rupture is often caused by some minor event. These ruptures are often associated with rotator cuff tears, especially in older people.

After lifting some weight boxes, a 45 year old man appeared bulging on the lower arm. He reported a history of sudden sharp pain on the upper arm, often accompanied by an audible clap. The male biceps tendon was diagnosed as ruptured and it was in stage 1 of the repair process. The rupture can be classified as a mild second degree sprain.

The patient is treated by intramuscular bolus injection of about 0.1U/kg to about 25U/kg of a neurotoxin into the biceps. Preferably, the neurotoxin is a botulinum toxin type E and/or F, more preferably a botulinum toxin type A. The particular dose and frequency of administration depends on a number of factors and is determined by the treating physician. The patient was also instructed to rest and compress the biceps with ice. The patient is able to curl within about three days after administration of the neurotoxin. After approximately three more days, the patient underwent a process of reduced inflammation, which is an indication that the patient entered stages 2 and 3 of the repair process. And the pain experienced by the patient is significantly reduced. Botulinum toxin type A can also be administered topically in amounts of about 10 units to about 200 units for long-term (2-4 months) treatment for muscle fixation and pain reduction.

Example 2

Disruption of extensor structures (mechanism)

The rupture of the extensor structure of the knee occurs in two ways: for younger patients, as a result of sudden or violent forces (e.g., jumping, lifting); for elderly patients, this is a result of relatively mild forces. In both cases, there may have been some initial cause (arching) before. This condition can affect elderly patients who are generally sedentary and suddenly increase their activity level, or who have some pre-existing or co-existing disease such as diabetes, rheumatoid arthritis, and other systemic inflammatory diseases or who have previously undergone knee surgery.

A 22 year old female soccer player was unable to extend his knees. The patient is not able to do a straight leg lift, but she can also walk if she places her hands on her thighs and keeps her knees extended. Plain radiography indicated that the patella was in a lower than normal position. The quadriceps muscle of the patient is diagnosed as severely ruptured.

After ascertaining that the injury is severe (three degrees), the patient agrees to undergo reconstructive surgery. After surgery, the patient is treated by intramuscular bolus injection of about 0.1U/kg to about 25U/kg of a neurotoxin, such as between about 10 units and about 400 units of a botulinum toxin type A, into the quadriceps muscle. Preferably the neurotoxin is botulinum toxin type A. The particular dose and frequency of administration depends on a number of factors and is determined by the treating physician. The patient was also instructed to rest and compress the quadriceps with ice. Within about 15 days after administration of the neurotoxin, the injured muscle may gradually exercise and move. The patient is then encouraged to gently exercise the recovering muscle to strengthen it and the surrounding muscles. As the toxin effect diminishes, the patient can then quickly engage in a physical treatment regimen or resume regular activities and/or exercise. If the patient lives on this activity, botulinum toxin treatment will help her return to this activity early. Prolonged (2-4 months) muscle fixation can also be performed using from about 10 units to about 200 units of botulinum toxin type A topically.

Example 3

Treatment of shin splints

Runners are often prone to shin splits in the lower extremities, which can cause pain and can limit such activity. Calf pain caused by a shin split is caused by a minor tear in the leg muscles at their contact point with the shin. There are two types: 1. an anterior shin clamp that occurs in the anterior portion of the tibia. 2. Posterior tibial clamp occurs along the inside (medial side) of the leg of the tibia.

The anterior shin clip is caused by muscle imbalance, insufficient cushioning or running with the toes. Excessive prone positioning has an effect on both the anterior and posterior shin clamps.

In treating a sprained muscle, such as a shin clip, five steps are suggested: (1) protecting the damaged muscle from further injury through the use of splints, pads and/or crutches; (2) the activity is limited, typically within 48 to 72 hours, to initiate the healing process. A short acting botulinum toxin type E or F or modified botulinum toxin type a can be administered to reduce the time of biological activity of type a toxin in vivo (even with a short period of flaccid muscle paralysis). Suitable botulinum toxins having a reduced time to biological activity in vivo suitable for use in the present invention, including botulinum toxin type A, are described in co-pending U.S. patent application Ser. No. 09/620,840, which is hereby incorporated by reference in its entirety. For more severe sprains, the restriction of activity may last from weeks to months. Because of the long-term restriction of activity, it is desirable to administer a botulinum toxin, such as the (unmodified) botulinum toxin type B, or more preferably botulinum toxin type A, with a longer duration of action. Without such treatment, the patient may experience weeks of activity limitation. As the healing process begins, it is contemplated that the affected muscles will be subjected to mild motion and movement; (3) ice compress should be used for 15-20 minutes per hour; (4) keeping the compression such as elastic bandage in the ice compress room; and (5) raising the damaged area to minimize swelling.

Example 4

Subcloning of BoNT/A-L chain Gene

This example describes a method for cloning a polynucleotide sequence encoding a BoNT/A-L chain. The DNA sequence encoding the BoNT/A-L chain was amplified by a PCR protocol using synthetic oligonucleotides having the sequences 5'-AAAGGCCTTTTGTTAATAAACAA-3' (SEQ ID #1) and 5'-GGAATTCTTACTTATTGTATCCTTTA-3' (SEQ ID # 2). The use of these primers allows the introduction of Stu I and EcoR I defined sites at the 5 'and 3' ends of the BoNT/A-L chain gene fragment, respectively. These restriction sites are then used to facilitate unidirectional subcloning of the amplification product. In addition, these primers introduce a stop codon at the C-terminus of the L chain coding sequence. In the amplification reaction, chromosomal DNA from c.botulinum (strain 63A) was used as a template.

The PCR amplification was performed using a PCR amplification system containing 10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl₂0.2mM of each deoxynucleotide triphosphate (dNTP), 50pmol of each primer, 200ng of genomic DNA, and 2.5 units of Taq-polymerase (Promega) in a volume of 100. mu.l. The reaction mixture was subjected to 35 denaturation (1 min at 94 ℃), annealing (2 min at 37 ℃) and polymerization (2 min at 72 ℃), andthe cycle of (2). Finally, the reaction was extended for another 5 minutes at 72 ℃.

The PCR amplification product was digested with Stu I and EcoR I, purified by agarose gel electrophoresis, and ligated into Sma I and EcoR I digested pBluescript IISK to give a plasmid, pSAL. Bacterial transformants harboring this plasmid were isolated by standard procedures. The identity of the L-strand polynucleotide of this clone was confirmed by double-stranded plasmid sequence analysis using SEQUENASE (United states biochemicals) according to the manufacturer's instructions. Synthetic oligonucleotide sequencing primers were prepared as needed to obtain overlapping sequencing protocols. The sequence of this clone was found to be similar to that of Binz et al, j.biol.chem.265: 9153(1990) and Thompson et al, Eur.J.biochem.189: 73(1990), the sequences disclosed in (A) and (B) are identical.

Site-directed mutants can also be made designed to mediate the enzymatic activity of the BoNT/A-L chain.

Example 5

Expression of botulinum toxin type A-L (BoNt/A-L) chain fusion proteins

This example describes a method to verify the expression of wild type L chain as a therapeutic component in bacteria harboring the pCA-L plasmid. A well-isolated bacterial flora harboring either pCAL was inoculated into L-broth containing 100. mu.g/ml ampicillin and 2% (w/v) glucose and cultured overnight at 30 ℃ with shaking. The overnight-cultured culture was diluted 1: 10 with fresh L-broth containing 100. mu.g/ml ampicillin and cultured for 2 hours. Fusion protein expression was induced by adding IPTG thereto to a final concentration of 0.1 mM. After further 4 hours of incubation at 30 ℃, the bacteria were harvested by centrifugation at 6,000 Xg for 10 minutes.

Small-scale SDS-PAGE analysis confirmed the presence of a 90kDa protein band in bacterial samples derived from IPTG-induction. Such M_rWith the predicted MBP (. about.40 kDa) and BoNT/A-L chain ((II))50kDa) component. Furthermore, the IPTG-induced clones comprise substantially greater amounts of fusion protein when compared to samples isolated from control cultures.

The presence of the desired fusion protein in IPTG-induced bacterial extracts can also be determined using Cenci di Bello et al in eur.j. biochem.219: the polyclonal anti-L chain probe described in 161(1993) was confirmed by Western blotting. Bands were visualized on PVDF membranes (Pharmacia; MiltonKeynes, UK) using anti-rabbit immunoglobulin (Bio-Rad; Heimel Hempstead, UK) conjugated to horseradish peroxidase and an ECL detection system (Amersham, UK). The results of the Western blot confirmed the presence of the predominant fusion protein and some weak bands corresponding to M compared to the intact fusion protein_rLow protein content. This observation suggests that the fusion protein undergoes limited degradation in bacteria or during isolation. This proteolytic cleavage was eliminated without the use of 1mM or 10mM benzamidine (Sigma; Poole, UK) during isolation.

The yield of the complete fusion protein isolated by the above method is still fully satisfactory for all the procedures described herein. Bacterial clones induced with IPTG produced 5-10mg of total MBP-wild type or mutated L chain fusion protein per liter of culture, based on the evaluation of the contaminating SDS-PAGE gels. Thus, the efficiency of the method of making a BoNT/A-L chain fusion protein described herein is high, despite the certainty that any limited proteolysis will occur.

The MBP-L chain fusion proteins encoded by the pCAL and pCAL-TyrU7 expression plasmids were purified from bacteria by amylose affinity chromatography. The recombinant wild-type or mutated L chain is then removed from the carbohydrate-binding domain of the fusion protein by using factor X₂Specific lysis was performed for isolation. This cleavage process can produce free MBP, free L chain and a small amount of uncleaved fusion protein. Although the resulting L chains present in such mixtures have been shown to have the desired activity, we have also performedAdditional purification steps. Thus, the mixture of cleavage products was applied to a second amylose affinity column, which bound MBP and uncleaved fusion protein. The free L chain is not retained on the affinity column and can therefore be isolated for use in the experiments described below.

Example 6

Purification of fusion proteins and isolation of recombinant BoNT/A-L chains

This example describes a method for preparing and purifying wild-type recombinant BoNT/a light chain from bacterial clones. The pellets from 1 liter of bacterial culture expressing wild-type BoNT/A-L chain protein were resuspended in column buffer [10mM Tris-HCl (pH8.0), 200mM NaCl, 1mM EGTA and 1mM DTT ] containing 1mM phenyl-methanesulfonyl fluoride (PMSF) and 10mM benzamidine]And dissolved by sonication. The lysate was made clear by centrifugation at 15,000 Xg for 15 minutes at 4 ℃. The supernatant was applied to an amylose affinity column (2X 10cm, 30ml resin)](New England BioLabs; Hitchin, UK). Unbound protein was washed off the resin with column buffer until the eluate was protein free, as judged by a stable absorbance reading at 280 nm. Subsequently, the bound MBP-L chain fusion protein was eluted with a column buffer containing 10mM maltose. The fractions containing the fusion proteins were combined and added with an additional 150mM NaCl, 2mM CaCl₂And 1mM DTT in 20mM Tris-HCl (pH8.0) at 4 ℃ for 72 hours.

In addition, 150mM NaCl and 2mM CaCl are added₂And 1mM DTT in 20mM Tris-HCl (pH8.0), the enzyme: using factor X at a substrate ratio of 1: 100₂(Promega; Southampton, UK) the fusion protein was cleaved. Dialyzed at 4 ℃ for 24 hours. The mixture of MBP and wild type or mutated L chain from this cleavage step was packed onto a 10ml amylose column equilibrated with column buffer. Preparing the flow-through fraction, etcAliquots were used for SDS-PAGE analysis to identify samples containing L chains. The remainder of the flow-through fraction was stored at-20 ℃. The total E.coli extract or purified protein was dissolved in SDS sample buffer and PAGE was performed according to standard methods. The results of this method indicate that the recombinant toxin fragment accounts for approximately 90% of the protein content of the sample.

The above results indicate that the methods described herein for preparing MBP-L chain fusion proteins can be used to efficiently prepare both wild-type and variant recombinant BoNT/A-L chains. Furthermore, the results also demonstrate that the recombinant L chain can be isolated from the maltose binding domain of the fusion protein and then purified.

Sensitive antibody-based assays were established to compare the enzymatic activity of recombinant L chain products and their natural counterparts. The assay uses antibodies specific for the intact C-terminal region of SNAP-25, which corresponds to the cleaved portion of BoNT/A. Western blotting of the product of the BoNT/A cleavage reaction of SNAP-25 indicated that the antibody was not able to bind to the sub-fragment of SNAP-25. Thus, the antibody heavy neurotoxin (reneurotoxin) used in the examples below can only detect intact SNAP-25. Loss of antibody binding can be used as an indicator of SNAP-25 proteolysis mediated by the addition of BoNT/A light chains or their recombinant derivatives.

Example 7

Assessment of proteolytic Activity of recombinant L chain on SNAP-25 substrate

This example describes a method to demonstrate that both native and recombinant BoNT/A-L chains can proteolytically hydrolyze a SNAP-25 substrate. A quantitative assay was used to compare the ability of wild-type and their recombinant analogs to cleave the SNAP-25 substrate. The substrate used in this experiment was obtained by preparing a glutathione-S-transferase (GST) -SNAP-25 fusion protein, which contains the cleavage site for thrombin, was expressed with the pGEX-2T vector and was obtained by using glutathionePeptide agarose affinity column chromatography for purification. Then, in the enzyme: the ratio of the substrates was 1: 100 in a reactor containing 150mM NaCl and 2.5mM CaCl₂In 50mM Tris-HCl (pH7.5), SNAP-25 was cleaved from the fusion protein using thrombin (Smith et al, Gene 67: 31 (1988)). The uncleaved fusion protein and the cleaved glutathione-binding domain bind to the gel. The recombinant SNAP-25 protein was eluted with the latter buffer and dialyzed against 100mM HEPES (pH7.5) at 4 ℃ for 24 hours. The total protein concentration is determined by conventional methods.

Rabbit polyclonal antibodies specific for the C-terminal region of SNAP-25 were raised against a synthetic peptide having the amino acid sequence-CANQRATKMLGSG (SEQ ID # 3). This peptide corresponds to residues 195 to 206 of synaptoblemin, and no N-terminal cysteine residue is found in native SNAP-25. Using maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as a cross-linked neurotoxin (Sigma; Poole, UK), the synthetic peptide was linked to Bovine Serum Albumin (BSA) (Sigma; Poole, UK) to improve antigenicity (Liu et al, Biochemistry 18: 690(1979) 1. affinity purification of anti-peptide antibodies was carried out using a column with antigenic peptides linked to aminoalkyl agarose resins through their N-terminal cysteine residues (Bio-Rad; Hemel Hempstead, UK), using cross-linked ethyl 3- (3-dimethylpropyl) carbodiimide, activated with iodoacetic acid, after successive washes of the column with a buffer containing 25 mM-HCl (pH 7.4) and 150mM NaCl, the peptide-specific antibodies were eluted with a solution of 100mM glycine (pH2.5) and 200mM Tris NaCl, and collected in a tube containing 0.2ml of 1M Tris-HCl (pH8.0) neutralization buffer.

Before evaluating its enzymatic activity, all recombinant preparations containing wild-type L chains were dialyzed overnight at 4 ℃ into 100mM HEPES (pH7.5) containing 0.02% Lubrol and 10. mu.M zinc acetate. Then, BoNT/A, previously reduced with 20mM DTT for 30 minutes at 37 ℃ and these dialysis samples were diluted to different concentrations with the latter HEPES buffer to which 1mM DTT was additionally added.

The reaction mixture included 5. mu.l of recombinant SNAP-25 substrate (final concentration of 8.5. mu.M) and 20. mu.l of reduced BoNT/A or recombinant wild-type L chain. All samples were incubated at 37 ℃ for 1 hour before treating the reaction with 25. mu.l of 2% aqueous trifluoroacetic acid (TFA) and 5mM EDTA (Foran et al, Biochemistry 33: 15365 (1994)). Aliquots of each sample for SDS-PAGE and Western blotting were prepared with polyclonal SNAP-25 antibody by adding SDS-PAGE sample buffer and boiling. The reactivity of the anti-SNAP-25 antibody was monitored with an ECL detection system and quantified by densitometric scanning.

The results of the Western blot clearly show the difference in proteolytic activity between the purified mutant L chain and the native or recombinant wild-type BoNT/A-L chain. Specifically, the recombinant wild-type L-strand can cleave the SNAP-25 substrate, although its potency will be somewhat lower than the reduced BoNT/a native L-strand used as a positive control in this method. Thus, enzymatically active forms of the BoNT/A-L chain can be produced by recombinant means and by subsequent isolation. In addition, a single amino acid substitution in the L chain protein may abolish the ability of the recombinant protein to degrade synaptic terminal proteins.

As with the previous biological Activity assay for wild-type recombinant BoNT/A-L chain, it was determined that MBP-L chain fusion proteins reduce Ga²⁺-the ability of activated catecholamines to be released from digitonin-pre-permeabilized bovine chromaffin cells. Consistently, the entire wild-type recombinant L chain fusion protein or the recombinant L chain fusion protein is bound by factor X₂Ga produced by cleavage to produce a wild-type recombinant L chain fusion protein comprising a mixture of free MBP and recombinant L chain²⁺The dose-dependent inhibition of stimulated release is equal to that produced by native BoNT/a.

Example 8

Reconstitution of native L chain, recombinant wild-type L chain and refined H chain

Native H and L chains were isolated from BoNT/A (List Biologicals Inc.; Campbell, U.S. A.) with 2M urea, reduced with 100mM DTT, and then purified according to established chromatographic methods (Kozaki et al, Japan J.Med.Sci.biol.34: 61 (1981); Maisey et al, Eur.J.biochem.177: 683 (1988)). The purified H chain is combined with natural L chain or recombinant wild-type L chain with equivalent molecular weight. Reconstitution was carried out by dialyzing the sample against a buffer consisting of 25mM Tris (pH8.0), 50. mu.M zinc acetate and 150mM NaCl at 4 ℃ for 4 days. After dialysis, the association of recombinant L chains and native H chains, which form disulfide-linked 150kDa duplexes, was monitored by SDS-PAGE and quantified by densitometric scanning. The proportion of double-stranded molecules formed with the recombinant L-chain is lower than that obtained when using the natural L-chain. Indeed, only about 30% of the recombinant wild-type or mutated L and H chains are reconstituted, while more than 90% of the native L and H chains are reconstituted. Despite the low efficiency of reconstitution, materials used in subsequent functional studies that bind sufficient recombinant L chains can be readily produced.

While the invention has been described with respect to various specific examples and embodiments, it will be understood that the invention is not so limited, and that it can be embodied in various forms without departing from the scope of the following claims. Other embodiments, variations and modifications are possible within the scope of the invention. For example, in accordance with the present disclosure, an injured muscle can be treated with between about 500 units and about 4,000 units of a botulinum toxin type B.

Claims (13)

1. A method of treating an injured muscle, the method comprising the step of topically administering a therapeutically effective amount of a neurotoxin to an injured muscle, thereby treating the injured muscle.

2. The method of claim 1, wherein the step of topically administering is intramuscular injection.

3. The method of claim 1, wherein the neurotoxin substantially immobilizes the damaged muscle.

4. The method of claim 1, wherein the neurotoxin is effective to immobilize the damaged muscle during stages 1 and 2 of the repair process of the damaged muscle.

5. The method of claim 1, wherein the neurotoxin is effective to immobilize the damaged muscle during phase 1 of the repair process of the damaged muscle.

6. The method of claim 1, wherein the neurotoxin is A, B, C₁Botulinum toxin type D, E, F or G.

7. The method of claim 1, wherein the neurotoxin is a recombinantly produced neurotoxin.

8. The method of claim 1, further comprising the step of treating the damaged muscle with physical therapy and/or surgery.

9. A method for treating an injured muscle, the method comprising the step of administering a therapeutically effective amount of a botulinum toxin locally in vivo to an injured muscle, whereby the injured muscle can be treated.

10. The method of claim 10, wherein the botulinum toxin is botulinum toxin type a.

11. A method for promoting healing of an injured muscle, the method comprising the step of administering a therapeutically effective amount of a botulinum toxin type a locally in vivo to the injured muscle, thereby promoting healing of the injured muscle.

12. A method for treating pain associated with an injured muscle, the method comprising the step of administering a therapeutically effective amount of a botulinum toxin locally in vivo to the injured muscle, thereby reducing pain associated with the injured muscle.

13. The method of claim 12, wherein the botulinum toxin is botulinum toxin type a.