US20020086036A1

US20020086036A1 - Methods for treating hyperhidrosis

Info

Publication number: US20020086036A1
Application number: US09/730,237
Authority: US
Inventors: Patricia Walker
Original assignee: Allergan Sales LLC
Current assignee: Allergan Sales LLC
Priority date: 2000-12-05
Filing date: 2000-12-05
Publication date: 2002-07-04
Also published as: US20120114703A1; US7479281B1; US8420105B2; US20100272754A1; US8025889B2; US20090041805A1

Abstract

Methods for treating hyperhidrosis is disclosed herein. In one embodiment, the method includes a step of administering a neurotoxin to a skin area to alleviate excessive sweating. In another embodiment, the method employs a needleless injector to affect the administration of a neurotoxin, for example botulinum toxin type A.

Description

BACKGROUND

In mammals, for example human beings, sweating is a normal thermoregulation process. Also, sweating is a normal physiological response to a psychological stress or emotional stimuli. For most people, sweating is only a minor cosmetic annoyance. For others, however, sweating may be excessive and become a socially or medically crippling handicap. The present invention relates to methods for treating excessive sweating in a mammal, including a human being, wherein the methods include a step of administering a neurotoxin to a mammal.

Hyperhidrosis is a disorder in which there is an exaggerated sweat secretion involving both the eccrine and the apocrine sweat glands. The excessive sweating usually occurs in the palms, soles, and axillae. Palmar hyperhidrosis is a condition of excessive sweating in the hand. Such condition may be socially embarrassing. Plantar hyperhidrosis is a condition of excessive sweating in the foot. This condition may cause blisters, infections, and bromohidrosis. Axillary hyperhidrosis is a condition of excessive sweating in the armpit. In axillary hyperhidrosis, as much as 26 mL/h of sweat can be excreted from each armpit. Such excessive sweating is not only socially embarrassing but may even cause staining and rotting of clothes.

Presently, the cause of hyperhidrosis is unknown. However, what is known is that the 3 to 4 million sweat glands of the body are under the control of the hypothalamus and the sympathetic system. Afferent impulses from sensors on the skin and other parts of the body travel to the hypothalamus, which integrates the information for chemoregulation of the body. The preoptic area of the anterior hypothalamus then sends efferent impulses via sympathetic fibers back out to the body. Segment T2 to T4 of the spinal chord innervate the head and neck area; fibers in segment T2 to T8 innervate the upper limbs; fibers in segment T6 to T10 innervate the trunk; and finally fibers in T11 to T12 innervate the lower extremities.

Although sympathetic innervations typically rely on adrenergic neurotransmitters, acetylcholine is the neurotransmitter released by the sympathetic nerve terminals involved in innervating the sweat glands. However, that is not to say that only acetylcholines can innervate the sweat glands. Some reports have shown that eccrine and apocrine glands respond to α- and β-adrenergic agonists as well.

Although the hypothalamus has a significant role in controlling the rate of sweating, other physical variables may affect the rate of sweat secretion. For example, sweating rate may also be affected by variables such as wetness and blood flow. Additionally, the rate of sweating varies greatly among people and is related to acclimatization, sex, age, and maybe even diet.

With respect to treating hyperhidrosis, various treatments are being used. For example, topical administration aluminum chloride is a common practice. It is thought that aluminum chloride mechanically obstruct eccrine sweat glands to reduce sweating, although some evidence shows that the reduction in sweat may result from atrophy of the secretory cells. A downside of using aluminum chloride is that the aluminum chloride may react with the water content of the sweat to form hydrochloric acid. The formation of hydrochloric acid may cause severe skin irritation.

Other topical preparations are also being used. For example, treatment of plantar and palmar hyperhidrosis includes use of glutaraldehyde and tannic acid (strong tea). However, this treatment may cause a browning of the skin.

Anticholinergics, both systemic and topical, are also being used. However, most patients cannot tolerate the side effects.

In addition to the described adverse effect of the above methods, the above treatment methods are effective to alleviate excessive sweating for only a brief duration of time, thus requiring frequent treatments, i.e. daily or weekly.

Surgical treatment involving sweat gland excission and sympathectomy may provide for a longer duration of alleviation from hyperhidrosis. However, these invasive treatments are rarely indicated due to the adverse consequences and cost. For example, surgery may cause contractures. Sympathectomy may result in complications including infection, pneumothorax, Horner's syndrome, resumption of sweating, and compensatory hyperhidrosis. Additionally, hyperhidrosis may resume after surgery or sympathectomy.

Botulinum Toxin

The anaerobic, gram positive bacterium 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.

BoNT/A is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) serotype A is a LD ₅₀in mice. One unit (U) of botulinum toxin is defined as the LD₅₀upon 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, C₁, D, E, F and G each of which is distinguished by neutralization with serotype-specific antibodies. The different serotypes of botulinum toxin 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 LD₅₀for 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 peripheral intramuscular BoNt/A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of BoNt/A averages about three months.

Although all the 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. For example, 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. Finally, botulinum toxin serotype C ₁(BoNT/C₁) 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.

Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, the toxin binds 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.

In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This last step is thought to be mediated by the amino end segment of the H chain, H _N, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin then translocates through the endosomal membrane into the cytosol.

The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the H and 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. Tetanus neurotoxin, botulinum toxin/B/D,/F, and/G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.

The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the BoNt/A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. BoNT/B and C ₁are apparently produced as only a 500 kD complex. BoNT/D is produced as both 300 kD and 500 kD complexes. Finally, BoNT/E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) 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.

In vitro studies have indicated that 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 ₁, 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. However, even 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. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy. Additionally, it is known that BoNt/B has, upon intramuscular injection, a shorter duration of activity and is also less potent than BoNt/A at the same dose level.

It has been reported that BoNt/A has been used in clinical settings as follows:

(1) about 75-125 units of BOTOX® ¹per intramuscular injection (multiple muscles) to treat cervical dystonia;

(2) 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);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincter injection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper lid and the lateral pre-tarsal orbicularis oculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 units of BOTOX®, the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).

(6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® into five different upper limb flexor muscles, as follows:

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

(b) flexor digitorum sublimus: 7.5 U to 30 U

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

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

(e) 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.

The success of BoNt/A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes. A study of two commercially available BoNT/A preparations (BOTOX® and Dysport®) and preparations of BoNT/B and F (both obtained from Wako Chemicals, Japan) has been carried out to determine local muscle weakening efficacy, safety and antigenic potential. Botulinum toxin preparations were injected into the head of the right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscle weakness was assessed using the mouse digit abduction scoring assay (DAS). ED ₅₀values were calculated from dose response curves. Additional mice were given intramuscular injections to determine LD₅₀doses. The therapeutic index was calculated as LD₅₀/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 (1.5 or 6.5 ng/kg for BoNt/B or 0.15 ng/kg for BOTOX®). Peak muscle weakness and duration were dose related for all serotypes. DAS ED₅₀values (units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, BoNt/B: 27.0 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: 3.2. 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. As shown by the therapeutic index values, 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. Furthermore, in rabbits, serotype B was more antigenic than was BOTOX®, possibly because of the higher protein load injected to achieve an effective dose of BoNt/B.

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

Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. 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 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 to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic and most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephine. In most instances acetylcholine has an excitatory effect. However, 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 postganglionic 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 postganglionic neurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic neurons of the parasympathetic nervous system, as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the synapses between the preganglionic and postganglionic 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. A wide variety of 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.

Botulinum toxin has been shown to be effective in treating hyperhidrosis. Especially, noteworthy is that botulinum toxin may alleviate hyperhidrosis for up to 11 months. For example, Odderson, Dermatol Surg (1988) 24:1237-1241, discloses that intracutaneous injections of botulinum toxin type A to the sweating area of the skin reduces excessive sweating; and Bushara et al., Clinical and Experimental Dermatology (1996) 21:276-278, disclose that subcutaneous injections of botulinum toxin type A can selectively denervate the local sweat glands to produce an anhidrotic patch. The alleviation from hyperhidrosis is up to 11 months.

The present methods for treating hyperhidrosis, for example those described by Odderson and Bushara et al., rely on the use of a needle, for example a 32 gauge needle, and a syringe for administration of a drug. Although the method of treatment is quite effective, this mode in administration often causes pain. Additionally, it is very difficult to deliver the right amount of drug to the right layer of the skin, for example the dermis layer. Consequently, an administration by a needle may result in non-specific delivery, which lessens the treatment efficacy. For example, misdirected injection into the epidermis or subcutaneous tissue is not efficacious. Furthermore, non-specific delivery may also causes adverse effects. For example, injection into the subcutaneous tissue may result in diffusion of the toxin into surrounding tissues. This non-specific diffusion of toxin may result in unwanted blockade of neuromuscular transmissions. Such neuromuscular transmission blockade may result in unwanted temporary loss of muscular functions, for example the hands. There remains a need for a method to treat hyperhidrosis more effectively.

SUMMARY

The methods of treating hyperhidrosis as described herein overcome at least one of the aforementioned problems.

In accordance with the invention, a method for treating hyperhidrosis in a mammal, for example a human being, includes the step of locally administering a drug particle to an affected skin area without using a needle. For example, a needleless injector may be used to affect a needleless administration or injection of the drug particle. For example, a needleless injector such as that sold by PowderJect, Oxford, UK, may be employed in accordance with this invention.

Further in accordance with the invention, the drug particle is administered to at least one layer of the skin, for example the epidermis layer, the dermis layer and/or the hypodermis layer. The dermis layer is believed to contain sweat glands and/or nerves innervating the sweat glands.

Still further in accordance with the invention, the drug particle is administered to at least one layer of the skin without substantially being administered to the muscle tissue beneath. The selective administration may be affected through the use of a needleless injector.

Still further in accordance with the invention, the drug particle comprises a neurotoxin. The neurotoxin may include a targeting component, a therapeutic component and a translocation component. The targeting component may bind to a presynaptic nerve terminal, for example a presynaptic nerve terminal of a cholinergic neuron. For example, the targeting component may include a carboxyl end segment of a heavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C ₁, D, E, F, G or a variant thereof. The therapeutic component may substantially interfere with the release of neurotransmitters from a neuron or its terminals. For example, the therapeutic component may include a light chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E, F, G or a variant thereof. The translocation component may facilitate the transfer of at least a part of the neurotoxin into the cytoplasm of the target cell. For example, the translocation component may include an amino end fragment of a heavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E, F, G or a variant thereof.

Still further in accordance with the invention, the neurotoxin is botulinum toxin type A. The neurotoxin may be produced recombinantly. For example, botulinum toxin type A may be produced recombinantly.

Still further in accordance with the invention, the drug particle may include a neurotoxin and a carrier, wherein the neurotoxin is coated onto the carrier. Any biologically compatible dense material such as gold, platinum and/or ice crystal may be used as a carrier. For example, a gold particle may be coated with a botulinum toxin type A to form a drug particle useful in this invention.

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

Definitions

Before proceeding to describe the present invention, the following definitions are provided and apply herein.

“Affected skin area” means an area with excessive sweating.

“Drug particle” means a drug, for example, a neurotoxin, alone or in combination with other matters, for example, gold.

“Without using a needle” means injecting a measurable amount of drug particle without the use of a standard needle.

“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.

“H _N” 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.

“H _C” 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.

“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.

“Neurotoxin” means a chemical entity that is capable of interfering with the functions of a neuron. The “neurotoxin” may be naturally occurring or man-made.

“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. For example, 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. Additionally, the biological effect of a variant may be decreased. For example, a variant light chain of a botulinum toxin type A having a leucine base motif removed may have a shorter biological persistence than that of the parent (or native) botulinum toxin type A light chain.

DETAILED DESCRIPTION OF THE INVENTION

Methods for treating hyperhidrosis in mammals, for example human beings, are disclosed herein. In one broad embodiment, a method of treating hyperhidrosis includes a step of locally administering a drug particle to an affected skin area without using a needle, wherein the drug particle is at least effective to reduce excessive sweating.

The skin has two distinct layers and varies in thickness from about 1.5 to about 4 mm or more, depending on the regions of the body. The first layer is the superficial layer called the epidermis. It is a relatively thick epithelium. Deep to the epidermis is the second layer called the dermis. The dermis is a fibrous connective tissue and comprises sweat glands and nerves, or nerve terminals, innervating such sweat glands.

Just deep to the skin lies a fatty layer called the hypodermis, which may also be considered a part of a subcutaneous layer. Beneath the hypodermis or subcutaneous layer lies the deep fascial investment of the specialized structures of the body, for example the muscles.

Accordingly, the method of this invention delivers the drug particle to at least one layer of the skin. Preferably, the drug is delivered to the layer of the skin in which the sweat glands and/or the nerve terminals innervating such sweat glands are found. For example, in a prefer embodiment, the drug particle is administered to the dermis layer. More preferably, the drug particle is administered to at least one layer of the skin and not substantially to any tissues beneath the skin. For example, in one embodiment, the administered drug particle is delivered to the dermis layer of the skin and not to the subcutaneous layer. In a preferred embodiment, the administered drug particle is delivered to the dermis layer of the skin and not to the muscle tissues beneath.

The administration of drug particles according to the invention may be affected through the use of a needleless injector, which is known in the art. For example, Bellhouse et al. in U.S. Pat. Nos. 6,053,889 ('889), 6,013,050 ('050), 6,010,478 ('478), 6,004,286 ('286) and 5,899,880 ('880) disclose novel needleless injectors. The disclosures therein are incorporated in their entirety by reference herein. In one embodiment, the needleless injector comprise an elongated tubular nozzle and is connected to or capable of connection to a suitable energizing means for producing a supersonic gas flow, for example a burst of helium, which accelerates fine drug particles to high velocity toward a skin surface and into the skin surface. Such a device may be purchased from PowderJect Pharmaceuticals, Oxford, UK. In a preferred embodiment, the gas pressure provided must be sufficient to discharge the drug particles into a targeted site, for example the dermis, but not so great as to damage the target. In another embodiment, the gas pressure provided is sufficient to deliver the drug particle to a target site, for example the dermis, but not so great as to damage the skin surface, for example the epithelium. In a more preferred embodiment, the gas pressure is sufficient to deliver the drug particle to the dermis layer, but not to the layers below, for example the subcutaneous layer and/or the muscle tissues. In an even more preferred embodiment, the gas pressure provided must be sufficient to discharge the drug particles into a targeted site, for example the dermis, but not so great as to (1) damage the skin surface and (2) deliver the drug particle to the muscle tissue.

Advantages for using a needleless injector for the treatment of hyperhidrosis according to the present invention include, for example, an optimal delivery to a specific skin layer, for example the dermis layer. Furthermore, in the case where the drug particle is delivered to the dermis and not the muscle tissues, the treatment does not cause a loss of motor function in the area being treated. Also, the use of a needleless injector according to the present invention improves clinical safety by eliminating the risk of infection from accidental injury with needles or from potential splash back of bodily fluids from liquid jet injectors, thereby avoiding the possibilities of cross-contamination of blood-borne pathogens such as HIV and hepatitis B. The needleless injector, such as the PowderJect System, also offers an optimal and specific delivery of drug particles to treat hyperhidrosis with no pain or skin damage such as bruising or bleeding.

In another broad embodiment, the drug particle comprises a neurotoxin. The neurotoxin may include a targeting component, a therapeutic component and a translocation component. The targeting component may bind to a presynaptic nerve terminal, for example a presynaptic nerve terminal of a cholinergic neuron. For example, the targeting component may include a carboxyl end segment of a heavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C ₁, D, E, F, G or a variant thereof. In a preferred embodiment, the targeting component comprises a carboxyl end segment of a heavy chain of a botulinum toxin type A.

The therapeutic component may substantially interfere with the release of neurotransmitters from a neuron or its terminals. For example, the therapeutic component may include a light chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C ₁, D, E, F, G or a variant thereof. In a preferred embodiment, the therapeutic component comprises a light chain of a botulinum toxin type A.

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

In one embodiment, the targeting component comprises a carboxyl end fragment of a heavy chain of a botulinum toxin type A, the therapeutic component comprises a light chain of a botulinum toxin type A and the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type A. In a preferred embodiment, the neurotoxin of the present invention comprises a botulinum toxin type A. For example, very useful botulinum toxin type A may be obtained from Allergan, Inc., under the trade name BOTOX®.

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

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

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

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

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

In one embodiment, the neurotoxin injected may be a nucleotide sequence. For example, the nucleotide sequence may be that of botulinum toxin type A (SEQ. ID. #1), type B (SEQ. ID. #2 and #3), type C ₁(SEQ. ID #4), type D (SEQ. ID. #5), type E (SEQ. ID. #6 and #7), type F (SEQ. ID. #8) and type G (SEQ. ID. #9), variants thereof or fragments thereof. Preferably, the nucleotide fragment injected can encode a therapeutic component, for example, a light chain.

A drug particle may comprise a neurotoxin alone or a neurotoxin in combination with other drugs and/or agents. In either case, the neurotoxin may be prepared as pharmaceutical compositions. The composition may contain one or more added materials such as carriers and/or excipients. As used herein, “carriers” and “excipients” generally refer to substantially inert, non-toxic materials that do not deleteriously interact with other components of the composition. These materials may be used to increase the amount of solids in particulate pharmaceutical compositions, such as to form a powder of drug particles suitable for use with a needleless injector. Examples of suitable carriers include water, silicone, gelatin, waxes, and the like. Although a naked nucleotide sequence may be injected in accordance with this invention, it is preferable that the injected nucleotide be accompanied by a carrier, for example See Felgner et al, U.S. Pat. No. 5,459,127, the disclosure of which is incorporated in its entirety herein by reference.

Other suitable carriers include any high density, biologically inert materials. For example, tungsten, platinum, iridium gold and/or ice crystal may be employed as carriers. In a preferred embodiment, the carrier is less than about 10 μm, more preferably less than about 5 μm, even more preferably less than about 3 μm. High density carriers of such size may readily enter living cells without unduly injuring such cells. In one embodiment, a drug particle comprises a neurotoxin, for example botulinum toxin type A, and a carrier, for example a high density material of less than 5 μm, wherein the neurotoxin is coated onto the high density carrier using techniques commonly known in the art. Ice crystals and gold are preferred carriers of this invention. Ice crystal particles are readily available in average sizes of 0.5 to 2.0 μm in diameter and are thus suited for intracellular delivery. Gold is also a preferred carrier, since gold has a high density and is relatively inert to biological materials and resists oxidation. Moreover, gold is readily available in the form of spheres having an average diameter of from about 0.2 to about 3 μm. In a preferred embodiment, neurotoxin is coated onto ice crystal and/or gold carriers to form drug particles. In a more preferred embodiment, botulinum toxin type A is coated onto ice crystal and/or gold carriers to form drug particles to be used in accordance with this invention.

Examples of normally employed “excipients,” include pharmaceutical grades of mannitol, sorbitol, inositol, dextrose, sucrose, lactose, trehalose, dextran, starch, cellulose, sodium or calcium phosphates, calcium sulfate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEG), and the like and combinations thereof. In one embodiment, the excipient may also include a charged lipid and/or detergent in the pharmaceutical compositions. Suitable charged lipids include, without limitation, phosphatidylcholines (lecithin), and the like. Detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant. Examples of suitable surfactants include, for example, Tergitol® and Triton® surfactants (Union Carbide Chemicals and Plastics, Danbury, Conn.), polyoxyethylenesorbitans, for example, TWEEN® surfactants (Atlas Chemical Industries, Wilmington, Del.), polyoxyethylene ethers, for example, Brij, pharmaceutically acceptable fatty acid esters, for example, lauryl sulfate and salts thereof (SDS), and the like. Such materials may be used as stabilizers and/or anti-oxidants. Additionally they may be used to reduce local irritation at the site of administration.

In one broad embodiment, the step of administering a drug particle according to the present invention may include other steps. These other steps may be carried out before, in conjunction with and/or after the step of administering the drug particle according to the invention. In one embodiment, these other steps may include applying topical medications, for example aluminum chloride; applying an iontophoresis procedure; administering anticholinergics orally or systemically. In severe hyperhidrosis, other steps may include surgical management and/or sympathectomy.

The following examples demonstrate how the various conditions of hyperhidrosis may be treated according to the present invention. Although particular doses are described, the dose administered can vary widely according to the severity of the condition and other various patient variables including size, weight, age, and responsiveness to therapy.

The examples also show how a neurotoxin or components thereof may be recombinantly synthesized and reconstituted. The examples relating to recombinant synthesis are substantially similar to the Examples of International Patent Application Publication WO 95/32738, the disclosure of which is incorporated in its entirety herein by reference.

EXAMPLE 1

Treatment of Gustatory Sweating [0085]
Gustatory sweating (Frey's syndrome, auriculotemporal syndrome) is sweating of the facial skin during meals and commonly is seen following parotid gland surgery and trauma to the preauricular region. Denervated sweat glands become reinnervated by misdirected sprouting of parasympathetic secretomotor fibers that have lost their “target organ,” the salivary gland. Gustatory sweating is experienced by 13-50% of patients after pariodectomy. [0086]
A 40 year old man presents with a classic case of Frey's syndrome. The area of hyperhidrosis on the face is visualized by means of an iodinestarch solution (Minor's iodine-starch test) after sweating is stimulated by having the patient chew an apple or sour fruit candy. The hyperhidrosis area is then marked with a pen. [0087]
Botulinum toxin type A coated on gold particle carrier is loaded into a needleless injector. The projection pressure is set so that the drug particles, i.e., the botulinum toxin A coated gold particles, may be delivered to the dermis layer of the skin. Also, such an amount of the drug particle is loaded so that about 20 U to about 60 U of botulinum toxin type A is delivered to 8×10 cm[0088] ²of the demarcated skin area. The particular dose of the neurotoxin and area of injection, as well as the frequency of toxin administrations depend upon a variety of factors to be determined by the treating physician, as previously set forth.
Seven days after treatment, the gustatory sweating is measured using the Minor's iodine test. The hyperhidrotic area shows about a 93% reduction. The reduction in gustatory sweating starts after about 72 hours and remains up to about 12 months. [0089]

EXAMPLE 2

Treatment of Axillary Hyperhidrosis [0090]
Axillary hyperhidrosis is a condition which may be socially and emotionally disturbing. It is a condition of excessive sweating, which may even cause staining and decaying of clothes. Initially, the treatment usually consists of topical application of antiperspirants containing aluminium salts and/or tanning agents. lontophoresis using special axillary electrodes are also employed in the treatment of axillary hyperhidrosis. Oral sedatives, tranquillizers or anticholinergic drugs are sometimes used as an adjunct. [0091]
If the medical treatment proves ineffective or produces unacceptable side-effect, removal of the axillary sweat glands by surgical excision or liposuction is the other current option. Surgery and liposuction, although often effective in controlling excessive sweating, are commonly complicated by infection, bleeding, scarring, loss of axillary hair, hypoaesthesia, pain due to nerve injury or entrapment and, occasionally, reinnervation of the residual glands and recurrence of hyperhidrosis. Denervation of sweat glands by sympathectomy is also effective but carries the risk of pneumothorax, Homer's syndrome and other complications. [0092]
A 35 year old office female dancer presents with a severe case of axillary hyperhidrosis. The area of hyperhidrosis under the forearm is visualized by means of an iodinestarch solution (Minor's iodine-starch test). The hyperhidrosis area is then marked with a pen. [0093]
Botulinum toxin type A coated on crystal ice particle carrier is loaded into a needleless injector. The projection pressure is set so that the drug particles, i.e., the botulinum toxin A coated ice crystal particles, may be delivered to the dermis layer of the skin. Also, such an amount of the drug particle is loaded so that about 20 U to about 60 of botulinum toxin type A is delivered to 8×15 cm[0094] ²of the demarcated skin area. The particular dose of the neurotoxin and area of injection, as well as the frequency of toxin administrations depend upon a variety of factors to be determined by the treating physician, as previously set forth.
Two weeks after treatment, the axillary sweating response is measured using the Minor's iodine test. The hyperhidrotic area shows about a 95% reduction. The reduction in axillary sweating remains up to about 27 months, preferably 11 months. [0095]

EXAMPLE 3

Treatment of Palmar Hyperhidrosis [0096]
Botulinum toxin has been injected into the palmar area to treat palmar hyperhidrosis, and has been found to be very effective. However, one of the main drawback of this treatment is the pain cause by the injection. The free nerve endings responsible for the pain sensation occur in the papillary dermis and epidermis whereas the sweat glands are imbedded deep in the dermis and in the upper layer of the subcutaneous tissue. To deliver the botulinum toxin as close to the sweat glands as possible, subdermal/subcutaneous injections would be optimal, and presumably less painful than more superficial injections. However, the deeper the injection the greater the risk of causing weakness of the small muscles of the hand and weakening the grip. [0097]
A 22 year old concert pianist presents with a palmar hyperhidrosis. The specific area of hyperhidrosis on the hand is visualized by means of an iodinestarch solution (Minor's iodine-starch test). The hyperhidrosis area is then marked with a pen. [0098]
Botulinum toxin type A coated on crystal ice particle carrier is loaded into a needleless injector. The projection pressure is set so that the drug particles, i.e., the botulinum toxin A coated ice crystal particles, may be delivered to the dermis layer of the skin. Also, such amount of the drug particle is loaded so that about 10 U to about 50 U of botulinum toxin type A is delivered to 10×15 cm[0099] ²of the demarcated skin area. An effective therapeutic dose of botulinum toxin is injected without substantial pain. Additionally, no substantial muscle weakness or fatigue of the hand is observed. The particular dose of the neurotoxin and area of injection, as well as the frequency of toxin administrations depend upon a variety of factors to be determined by the treating physician, as previously set forth.
Two weeks after treatment, the reduced sweating response is measured in the area of hyperhidrosis using the Minor's iodine test. The hyperhidrotic area shows about a 95% reduction. The reduction in sweating remains up to about 12 months. [0100]

EXAMPLE 4

Subcloning the BONT/A-L Chain Gene [0101]
This Example describes the methods to clone the polynucleotide sequence encoding the BoNT/A-L chain. The DNA sequence encoding the BoNT/A-L chain is amplified by a PCR protocol that employs synthetic oligonucleotides having the sequences, 5′-AAAGGCCTTTTGTTMTAAACAA-3′ (SEQ ID#10) and 5′-GGAATTCTTACTTATTGTATCCTTTA-3′ (SEQ ID#11). Use of these primers allows the introduction of Stu I and EcoR I restriction sites into the 5′ and 3′ ends of the BoNT/A-L chain gene fragment, respectively. These restriction sites are subsequently used to facilitate unidirectional subcloning of the amplification products. Additionally, these primers introduce a stop codon at the C-terminus of the L chain coding sequence. Chromosomal DNA from [0102] C. botulinum (strain 63 A) serves as a template in the amplification reaction.
The PCR amplification is performed in a 100 μl volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl[0103] 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 [0104] J. Biol. Chem. 265:9153 (1990), and Thompson et al., in Eur. J. Biochem. 189:73 (1990).
Site-directed mutants designed to compromise the enzymatic activity of the BoNT/A-L chain can also be created. [0105]

EXAMPLE 5

Expression of the Botulinum Toxin Type A-L (BoNt/VA-L) Chain Fusion Proteins [0106]
This Example describes the methods to verify expression of the wild-type L chains, which may serve as a therapeutic component, in bacteria harboring the pCA-L plasmids. Well isolated bacterial colonies harboring either pCAL are used to inoculate L-broth containing 100 μg/ml ampicillin and 2% (w/v) glucose, and grown overnight with shaking at 30° C. The overnight cultures are diluted 1:10 into fresh L-broth containing 100 μg/ml of ampicillin and incubated for 2 hours. Fusion protein expression is induced by addition of IPTG to a final concentration of 0.1 mM. After an additional 4 hour incubation at 30° C., bacteria are collected by centrifugation at 6,000×g for 10 minutes. [0107]
A small-scale SDS-PAGE analysis confirmed the presence of a 90 kDa protein band in samples derived from IPTG-induced bacteria. This M[0108] _ris consistent with the predicted size of a fusion protein having MBP (˜40 kDa) and BoNT/A-L chain (˜50 kDa) components. Furthermore, when compared with samples isolated from control cultures, the IPTG-induced clones contained substantially larger amounts of the fusion protein.
The presence of the desired fusion proteins in IPTG-induced bacterial extracts is also confirmed by Western blotting using the polyclonal anti-L chain probe described by Cenci di Bello et al., in [0109] Eur. J. Biochem. 219:161 (1993). Reactive bands on PVDF membranes (Pharmacia; Milton Keynes, UK) are visualized using an anti-rabbit immunoglobulin conjugated to horseradish peroxidase (Bio-Rad; Hemel Hempstead, UK) and the ECL detection system (Amersham, UK). Western blotting results confirmed the presence of the dominant fusion protein together with several faint bands corresponding to proteins of lower M_rthan the fully sized fusion protein. This observation suggested that limited degradation of the fusion protein occurred in the bacteria or during the isolation procedure. Neither the use of 1 mM nor 10 mM benzamidine (Sigma; Poole, UK) during the isolation procedure eliminated this proteolytic breakdown.
The yield of intact fusion protein isolated by the above procedure remained fully adequate for ell procedures described herein. Based on estimates from stained SDS-PAGE gels, the bacterial clones induced with IPTG yielded 5-10 mg of total MBP-wild-type or mutant L chain fusion protein per liter of culture. Thus, the method of producing BoNT/A-L chain fusion proteins disclosed herein is highly efficient, despite any limited proteolysis that did occur. [0110]
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[0111] 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.

EXAMPLE 6

Purification of Fusion Proteins and Isolation of Recombinant BoNT/A-L Chains [0112]
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-HCl (pH 8.0), 200 mM NaCl, 1 mM EGTA and 1 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×g for 15 minutes at 4° C. Supernatants are applied to an amylose affinity column [2×10 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-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM, CaCI[0113] ₂and 1 mM DTT for 72 hours at 4° C.
Fusion proteins are cleaved with Factor X[0114] ₂(Promega; Southampton, UK) at an enzyme:substrate ratio of 1:100 while dialyzing against a buffer of 20 mM Tris-HCl (pH 8.0) supplemented with 150 mM NaCl, 2 mM, CaCl₂and 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. Remaining portions of the flow through fractions are stored at −20° C. Total E. coli extract or the purified proteins are solubilized in SDS sample buffer and subjected to PAGE according to standard procedures. Results of this procedure indicated the recombinant toxin fragment accounted for roughly 90% of the protein content of the sample.
The foregoing results indicates that the approach to creating MBP-L chain fusion proteins described herein could be used to efficiently produce wild-type and mutant recombinant BoNT/A-L chains. Further, the results demonstrate that recombinant L chains could be separated from the maltose binding domains of the fusion proteins and purified thereafter. [0115]
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. Thus, the antibody reagent 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. [0116]

EXAMPLE 7

Evaluation of the Proteolytic Activities of Recombinant L Chains Against a SNAP-25 Substrate [0117]
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. The SNAP-25 is then cleaved from the fusion protein using thrombin in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and 2.5 mM CaCl[0118] 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.
Rabbit polyclonal antibodies specific for the C-terminal region of SNAP-25 are raised against a synthetic peptide having the amino acid sequence, CANQRATKMLGSG (SEQ ID#12). 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 agent (Sigma; Poole, UK) to improve antigenicity (Liu et al., [0119] 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. After successive washes of the column with a buffer containing 25 mM Tris-HCl (pH 7.4) and 150 mM NaCl, the peptide-specific antibodies are eluted using a solution of 100 mM glycine (pH 2.5) and 200 mM NaCl, and collected in tubes containing 0.2 ml of 1 M Tris-HCl (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 μM 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. [0120]
Reaction mixtures include 5 μl recombinant SNAP-25 substrate (8.5 μM final concentration) and either 20 μl 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 μl aqueous 2% trifluoroacetic acid (TFA) and 5 mM EDTA (Foran et al., [0121] 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.
Western blotting results indicate clear differences between the proteolytic activities of the purified mutant L chain and either native or recombinant wild-type BoNT/A-L chain. Specifically, recombinant wild-type L chain cleaves the SNAP-25 substrate, though somewhat less efficiently than the reduced BoNT/A native L chain that serves as the positive control in the procedure. Thus, an enzymatically active form of the BoNT/A-L chain is produced by recombinant means and subsequently isolated. Moreover, substitution of a single amino acid in the L chain protein abrogated the ability of the recombinant protein to degrade the synaptic terminal protein. As a preliminary test of the biological activity of the wild-type recombinant BoNT/A-L chain, the ability of the MBP-L chain fusion protein to diminish Ca[0122] ²⁺-evoked catecholamine release from digitonin-permeabilized bovine adrenochromaffin cells is examined. Consistently, wild-type recombinant L chain fusion protein, either intact or cleaved with Factor X₂to produce a mixture containing free MBP and recombinant L chain, induced a dose-dependent inhibition of Ca²⁺-stimulated release equivalent to the inhibition caused by native BoNT/A.

EXAMPLE 8

Reconstitution of Native L Chain, Recombinant Wild-type L Chain with Purified H Chain [0123]
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., [0124] 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 μM zinc acetate and 150 mM NaCl 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.
While this invention has been described with respect to various specific examples and embodiments, it is to be understood that the invention is not limited thereto and that it can be variously practiced with the scope of the following claims. Other embodiments, versions, and modifications within the scope of the present invention are possible. [0125]
1 12 1 3891 DNA botulinum toxin 1 atgcaatttg ttaataaaca atttaattat aaagatcctg taaatggtgt tgatattgct 60 tatataaaaa ttccaaatgt aggacaaatg caaccagtaa aagcttttaa aattcataat 120 aaaatatggg ttattccaga aagagataca tttacaaatc ctgaagaagg agatttaaat 180 ccaccaccag aagcaaaaca agttccagtt tcatattatg attcaacata tttaagtaca 240 gataatgaaa aagataatta tttaaaggga gttacaaaat tatttgagag aatttattca 300 actgatcttg gaagaatgtt gttaacatca atagtaaggg gaataccatt ttggggtgga 360 agtacaatag atacagaatt aaaagttatt gatactaatt gtattaatgt gatacaacca 420 gatggtagtt atagatcaga agaacttaat ctagtaataa taggaccctc agctgatatt 480 atacagtttg aatgtaaaag ctttggacat gaagttttga atcttacgcg aaatggttat 540 ggctctactc aatacattag atttagccca gattttacat ttggttttga ggagtcactt 600 gaagttgata caaatcctct tttaggtgca ggcaaatttg ctacagatcc agcagtaaca 660 ttagcacatg aacttataca tgctggacat agattatatg gaatagcaat taatccaaat 720 agggttttta aagtaaatac taatgcctat tatgaaatga gtgggttaga agtaagcttt 780 gaggaactta gaacatttgg gggacatgat gcaaagttta tagatagttt acaggaaaac 840 gaatttcgtc tatattatta taataagttt aaagatatag caagtacact taataaagct 900 aaatcaatag taggtactac tgcttcatta cagtatatga aaaatgtttt taaagagaaa 960 tatctcctat ctgaagatac atctggaaaa ttttcggtag ataaattaaa atttgataag 1020 ttatagaaaa tgttaacaga gatttagaca gaggataatt ttgttaagtt ttttaaagta 1080 cttaacagaa aaacatattt gaattttgat aaagccgtat ttaagataaa tatagtacct 1140 aaggtaaatt acacaatata tgatggattt aatttaagaa atacaaattt agcagcaaac 1200 tttaatggtc aaaatacaga aattaataat atgaatttta ctaaactaaa aaattttact 1260 ggattgtttg aattttataa gttgctatgt gtaagaggga taataacttc taaaactaaa 1320 tcattagata aaggatacaa taaggcatta aatgatttat gtatcaaagt taataattgg 1380 gacttgtttt ttagtccttc agaagataat tttactaatg atctaaataa aggagaagaa 1440 attacatctg atactaatat agaagcagca gaagaaaata ttagtttaga tttaatacaa 1500 caatattatt taacctttaa ttttgataat gaacctgaaa atatttcaat agaaaatctt 1560 tcaagtgaca ttataggcca attagaactt atgcctaata tagaaagatt tcctaatgga 1620 aaaaagtatg agttagataa atatactatg ttccattatc ttcgtgctca agaatttgaa 1680 catggtaaat ctaggattgc tttaacaaat tctgttaacg aagcattatt aaatcctagt 1740 cgtgtttata catttttttc ttcagactat gtaaagaaag ttaataaagc tacggaggca 1800 gctatgtttt taggctgggt agaacaatta gtatatgatt ttaccgatga aactagcgaa 1860 gtaagtacta cggataaaat tgcggatata actataatta ttccatatat aggacctgct 1920 ttaaatatag gtaatatgtt atataaagat gattttgtag gtgctttaat attttcagga 1980 gctgttattc tgttagaatt tataccagag attgcaatac ctgtattagg tacttttgca 2040 cttgtatcat atattgcgaa taaggttcta accgttcaaa caatagataa tgctttaagt 2100 aaaagaaatg aaaaatggga tgaggtctat aaatatatag taacaaattg gttagcaaag 2160 gttaatacac agattgatct aataagaaaa aaaatgaaag aagctttaga aaatcaagca 2220 gaagcaacaa aggctataat aaactatcag tataatcaat atactgagga agagaaaaat 2280 aatattaatt ttaatattga tgatttaagt tcgaaactta atgagtctat aaataaagct 2340 atgattaata taaataaatt tttgaatcaa tgctctgttt catatttaat gaattctatg 2400 atcccttatg gtgttaaacg gttagaagat tttgatgcta gtcttaaaga tgcattatta 2460 aagtatatat atgataatag aggaacttta attggtcaag tagatagatt aaaagataaa 2520 gttaataata cacttagtac agatatacct tttcagcttt ccaaataggt agataatcaa 2580 agattattat ctacatttac tgaatatatt aagaatatta ttaatacttc tatattgaat 2640 ttaagatatg aaagtaatca tttaatagac ttatctaggt atgcatcaaa aataaatatt 2700 ggtagtaaag taaattttga tccaatagat aaaaatcaaa ttcaattatt taatttagaa 2760 agtagtaaaa ttgaggtaat tttaaaaaat gctattgtat ataatagtat gtatgaaaat 2820 tttagtacta gcttttggat aagaattcct aagtatttta acagtataag tctaaataat 2880 gaatatacaa taataaattg tatggaaaat aattcaggat ggaaagtatc acttaattat 2940 ggtgaaataa tctggacttt acaggatact caggaaataa aacaaagagt agtttttaaa 3000 tagagtcaaa tgattaatat atcagattat ataaacagat ggatttttgt aactatcact 3060 aataatagat taaataactc taaaatttat ataaatggaa gattaataga tgaaaaacca 3120 atttcaaatt taggtaatat tcatgctagt aataatataa tgtttaaatt agatggttgt 3180 agagatacac atagatatat ttggataaaa tattttaatc tttttgataa ggaattaaat 3240 gaaaaagaaa tcaaagattt atatgataat caatcaaatt caggtatttt aaaagacttt 3300 tggggtgatt atttacaata tgataaacca tagtatatgt taaatttata tgatccaaat 3360 aaatatgtcg atgtaaataa tgtaggtatt agaggttata tgtatcttaa agggcctaga 3420 ggtagcgtaa tgactacaaa catttattta aattcaagtt tgtatagggg gacaaaattt 3480 attataaaaa aatatgcttc tggaaataaa gataatattg ttagaaataa tgatcgtgta 3540 tatattaatg tagtagttaa aaataaagaa tataggttag ctactaatgc atcacaggca 3600 ggcgtagaaa aaatactaag tgcattagaa atacctgatg taggaaatct aagtcaagta 3660 gtagtaatga agtcaaaaaa tgatcaagga ataacaaata aatgcaaaat gaatttacaa 3720 gataataatg ggaatgatat aggctttata ggatttcatc agtttaataa tatagctaaa 3780 ctagtagcaa gtaattggta taatagacaa atagaaagat ctagtaggac tttgggttgc 3840 tcatgggaat ttattcctgt agatgatgga tggggagaaa ggccactgta a 3891 2 3876 DNA botulinum toxin 2 atgccagtta caataaataa ttttaattat aatgatccta ttgataatga caatattatt 60 atgatggaac ctccatttgc aaggggtacg gggagatatt ataaagcttt taaaatcaca 120 gatcgtattt ggataatacc cgaaagatat acttttggat ataaacctga ggattttaat 180 aaaagttccg gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttacttaaat 240 accaatgata aaaagaatat atttttccaa acattgatca agttatttaa tagaatcaaa 300 tcaaaaccat tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 360 gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 420 ttaattagta atccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480 tttggacctg ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 540 tttgcatcaa gggaaggctt tgggggtata atgcaaatga aattttgtcc agaatatgta 600 agcgtattta ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 660 ttttcagatc cagccttgat attaatgcat gaacttatac atgttttgca tggattatat 720 ggcattaaag tagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780 acagatacta tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 840 tctccttcta cagataaaag tatctatgat aaagttttgc aaaattttag ggggatagtt 900 gatagactta acaaggtttt agtttgcata tcagatccta acattaacat taatatatat 960 aaaaataaat ttaaagataa atataaattc gttgaagatt ctgaaggaaa atatagtata 1020 gatgtagaaa gtttcaataa attatataaa agcttaatgt taggttttac agaaattaat 1080 atagcagaaa attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1140 gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1200 tctgataaaa atatgggaaa agaatatagg ggtcagaata aagctataaa taaacaagct 1260 tatgaagaaa tcagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1320 aaagttccag gaatatgtat tgatgtcgat aatgaaaatt tgttctttat agctgataaa 1380 aatagttttt cagatgattt atctaaaaat gaaagagtag aatataatac acagaataat 1440 tatataggaa atgactttcc tataaatgaa ttaattttag atactgattt aataagtaaa 1500 atagaattac caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1560 tatgaaaaac aacccgctat aaaaaaagtt tttacagatg aaaataccat ctttcaatat 1620 ttatactctc agacatttcc tctaaatata agagatataa gtttaacatc ttcatttgat 1680 gatgcattat tagtttctag caaagtttat tcattttttt ctatggatta tattaaaact 1740 gctaataaag tagtagaagc aggattattt gcaggttggg tgaaacagat agtagatgat 1800 tttgtaatcg aagctaataa aagcagtact atggataaaa ttgcagatat atctctaatt 1860 gttccttata taggattagc tttaaatgta ggagatgaaa cagctaaagg aaattttgaa 1920 agtgcttttg agattgcagg atccagtatt ttactagaat ttataccaga acttttaata 1980 cctgtagttg gagtcttttt attagaatca tatattgaca ataaaaataa aattattaaa 2040 acaatagata atgctttaac taaaagagtg gaaaaatgga ttgatatgta gggattaata 2100 gtagcgcaat ggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2160 aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacaa atataatata 2220 tattctgaag aggaaaagtc aaatattaac atcaatttta atgatataaa ttctaaactt 2280 aatgatggta ttaaccaagc tatggataat ataaatgatt ttataaatga atgttctgta 2340 tcatatttaa tgaaaaaaat gattccatta gctgtaaaaa aattactaga ctttgataat 2400 actctcaaaa aaaatttatt aaattatata gatgaaaata aattatattt aattggaagt 2460 gtagaagatg aaaaatcaaa agtagataaa tacttgaaaa ccattatacc atttgatctt 2520 tcaacgtatt ctaatattga aatactaata aaaatattta ataaatataa tagcgaaatt 2580 ttaaataata ttatcttaaa tttaagatat agagataata atttaataga tttatcagga 2640 tatggagcaa aggtagaggt atatgatggg gtcaagctta atgataaaaa tcaatttaaa 2700 ttaactagtt cagcagatag taagattaga gtcactcaaa atcagaatat tatatttaat 2760 agtatgttcc ttgattttag cgttagcttt tggataagga tacctaaata taggaatgat 2820 gatatacaaa attatattca taatgaatat acgataatta attgtatgaa aaataattca 2880 ggctggaaaa tatctattag gggtaatagg ataatatgga ccttaattga tataaatgga 2940 aaaaccaaat cagtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3000 agatggtttt ttgtaactat tactaataat ttggataatg ctaaaattta tattaatggc 3060 acgttagaat caaatatgga tattaaagat ataggagaag ttattgttaa tggtgaaata 3120 acatttaaat tagatggtga tgtagataga acacaattta tttggatgaa atattttagt 3180 atttttaata cgcaattaaa tcaatcaaat attaaagaga tatataaaat tcaatcatat 3240 agcgaatagt taaaagattt ttggggaaat cctttaatgt ataataaaga atattatatg 3300 tttaatgcgg ggaataaaaa ttcatatatt aaactagtga aagattcatc tgtaggtgaa 3360 atattaatac gtagcaaata taatcagaat tccaattata taaattatag aaatttatat 3420 attggagaaa aatttattat aagaagagag tcaaattctc aatctataaa tgatgatata 3480 gttagaaaag aagattatat acatctagat ttggtacttc accatgaaga gtggagagta 3540 tatgcctata aatattttaa ggaacaggaa gaaaaattgt ttttatctat tataagtgat 3600 tctaatgaat tttataagac tatagaaata aaagaatatg atgaacagcc atcatatagt 3660 tgtcagttgc tttttaaaaa agatgaagaa agtactgatg atataggatt gattggtatt 3720 catcgtttct aggaatctgg agttttacgt aaaaagtata aagattattt ttgtataagt 3780 aaatggtagt taaaagaggt aaaaaggaaa ccatataagt caaatttggg atgtaattgg 3840 cagtttattc ctaaagatga agggtggact gaataa 3876 3 3876 DNA botulinum toxin 3 atgccagtta caataaataa ttttaattat aatgatccta ttgataataa taatattatt 60 atgatggagc ctccatttgc gagaggtacg gggagatatt ataaagcttt taaaatcaca 120 gatcgtattt ggataatacc ggaaagatat acttttggat ataaacctga ggattttaat 180 aaaagttccg gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttagttaaat 240 actaatgata aaaagaatat atttttacaa acaatgatca agttatttaa tagaatcaaa 300 tcaaaaccat tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga 360 gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac tgttaataaa 420 ttaatcagta atccaggaga agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480 tttggacctg ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat 540 tttgcatcaa gggaaggctt cgggggtata atgcaaatga agttttgccc agaatatgta 600 agcgtattta ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat 660 ttttcagatc cagccttgat attaatgcat gaacttatac atgttttaca tggattatat 720 ggcattaaag tagatgattt accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780 acagatgcta tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata 840 actccttcta cggataaaag tatctatgat aaagttttgc aaaattttag agggatagtt 900 gatagactta acaaggtttt agtttgcata tcagatccta acattaatat taatatatat 960 aaaaataaat ttaaagataa atataaattc gttgaagatt ctgagggaaa atatagtata 1020 gatgtagaaa gttttgataa attatataaa agcttaatgt ttggttttac agaaactaat 1080 atagcagaaa attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca 1140 gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg gtttaatata 1200 tctgataaag atatggaaaa agaatataga ggtcagaata aagctataaa taaacaagct 1260 tatgaagaaa ttagcaagga gcatttggct gtatataaga tacaaatgtg taaaagtgtt 1320 aaagctccag gaatatgtat tgatgttgat aatgaagatt tgttctttat agctgataaa 1380 aatagttttt cagatgattt atctaaaaac gaaagaatag aatataatac acagagtaat 1440 tatatagaaa atgacttccc tataaatgaa ttaattttag atactgattt aataagtaaa 1500 atagaattac caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta 1560 tatgaaaaac aacccgctat aaaaaaaatt tttacagatg aaaataccat ctttcaatat 1620 ttatagtctc agacatttct cttagatata agagatataa gtttaacatc ttcatttgat 1680 gatgcattat tattttctaa caaagtttat tcattttttt ctatggatta tattaaaact 1740 gctaataaag tggtagaagc aggattattt gcaggttggg tgaaacagat agtaaatgat 1800 tttgtaatcg aagctaataa aagcaatact atggataaaa ttgcagatat atctctaatt 1860 gttccttata taggattagc tttaaatgta ggaaatgaaa cagctaaagg aaattttgaa 1920 aatgcttttg agattgcagg agccagtatt ctactagaat ttataccaga acttttaata 1980 cctgtagttg gagccttttt attagaatca tatattgaca ataaaaataa aattattaaa 2040 acaatagata atgctttaac taaaagaaat gaaaaatgga gtgatatgta gggattaata 2100 gtagcgcaat ggctctcaac agttaatact caattttata caataaaaga gggaatgtat 2160 aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacag atataatata 2220 tattctgaaa aagaaaagtc aaatattaac atcgatttta atgatataaa ttctaaactt 2280 aatgagggta ttaaccaagc tatagataat ataaataatt ttataaatgg atgttctgta 2340 tcatatttaa tgaaaaaaat gattccatta gctgtagaaa aattactaga ctttgataat 2400 actctcaaaa aaaatttgtt aaattatata gatgaaaata aattatattt gattggaagt 2460 gcagaatatg aaaaatcaaa agtaaataaa tagttgaaaa ccattatgcc gtttgatctt 2520 tcaatatata ccaatgatac aatactaata gaaatgttta ataaatataa tagcgaaatt 2580 ttaaataata ttatcttaaa tttaagatat aaggataata atttaataga tttatcagga 2640 tatggggcaa aggtagaggt atatgatgga gtcgagctta atgataaaaa tcaatttaaa 2700 ttaactagtt cagcaaatag taagattaga gtgactcaaa atcagaatat catatttaat 2760 agtgtgttcc ttgattttag cgttagcttt tggataagaa tacctaaata taagaatgat 2820 ggtatacaaa attatattca taatgaatat acaataatta attgtatgaa aaataattcg 2880 ggctggaaaa tatctattag gggtaatagg ataatatgga ctttaattga tataaatgga 2940 aaaaccaaat cggtattttt tgaatataac ataagagaag atatatcaga gtatataaat 3000 agatggtttt ttgtaactat tactaataat ttgaataacg ctaaaattta tattaatggt 3060 aagctagaat caaatacaga tattaaagat ataagagaag ttattgctaa tggtgaaata 3120 atatttaaat tagatggtga tatagataga acacaattta tttggatgaa atatttcagt 3180 atttttaata cggaattaag tcaatcaaat attgaagaaa gatataaaat tcaatcatat 3240 agcgaatatt taaaagattt ttggggaaat cctttaatgt agaataaaga atattatatg 3300 tttaatgcgg ggaataaaaa ttcatatatt aaactaaaga aagattcacc tgtaggtgaa 3360 attttaacac gtagcaaata taatcaaaat tctaaatata taaattatag agatttatat 3420 attggagaaa aatttattat aagaagaaag tcaaattctc aatctataaa tgatgatata 3480 gttagaaaag aagattatat atatctagat ttttttaatt taaatcaaga gtggagagta 3540 tatacctata aatattttaa gaaagaggaa gaaaaattgt ttttagctcc tataagtgat 3600 tctgatgagt tttagaatac tatacaaata aaagaatatg atgaacagcc aacatatast 3660 tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggatt gattggtatt 3720 catcgtttct aggaatctgg aattgtattt gaagagtata aagattattt ttgtataagt 3780 aaatggtagt taaaagaggt aaaaaggaaa ccatataatt taaaattggg atgtaattgg 3840 cagtttattc ctaaagatga agggtggact gaataa 3876 4 3876 DNA botulinum toxin 4 atgccaataa caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 60 tatttagata ctcatttaaa tacactagct aatgagcctg aaaaagcctt tcgcattaca 120 ggaaatatat gggtaatacc tgatagattt tcaagaaatt ctaatccaaa tttaaataaa 180 cctcctcgag ttacaagccc taaaagtggt tattatgatc ctaattattt gagtactgat 240 tctgacaaag atacattttt aaaagaaatt ataaagttat ttaaaagaat taattctaga 300 gaaataggag aagaattaat atatagactt tcgacagata taccctttcc tgggaataac 360 aatactccaa ttaatacttt tgattttgat gtagatttta acagtgttga tgttaaaact 420 agacaaggta acaactgggt taaaactggt agcataaatc ctagtgttat aataactgga 480 cctagagaaa acattataga tccagaaact tctacgttta aattaactaa caatactttt 540 gcggcacaag aaggatttgg tgctttatca ataatttcaa tatcacctag atttatgcta 600 acatatagta atgcaactaa tgatgtagga gagggtagat tttctaagtc tgaattttgc 660 atggatccaa tactaatttt aatgcatgaa cttaatcatg caatgcataa tttatatgga 720 atagctatac caaatgatca aacaatttca tctgtaacta gtaatatttt ttattctcaa 780 tataatgtga aattagagta tgcagaaata tatgcatttg gaggtccaac tatagacctt 840 attcctaaaa gtgcaaggaa atattttgag gaaaaggcat tggattatta tagatctata 900 gctaaaagac ttaatagtat aactactgca aatccttcaa gctttaataa atatataggg 960 gaatataaac agaaacttat tagaaagtat agattcgtag tagaatcttc aggtgaagtt 1020 acagtaaatc gtaataagtt tgttgagtta tataatgaac ttacacaaat atttacagaa 1080 tttaactagg ctaaaatata taatgtacaa aataggaaaa tatatctttc aaatgtatat 1140 actccggtta cggcgaatat attagacgat aatgtttatg atatacaaaa tggatttaat 1200 atacctaaaa gtaatttaaa tgtactattt atgggtcaaa atttatctcg aaatccagca 1260 ttaagaaaag tcaatcctga aaatatgctt tatttattta caaaattttg tcataaagca 1320 atagatggta gatcattata taataaaaca ttagattgta gagagctttt agttaaaaat 1380 actgacttac cctttatagg tgatattagt gatgttaaaa ctgatatatt tttaagaaaa 1440 gatattaatg aagaaactga agttatatac tatccggaca atgtttcagt agatcaagtt 1500 attctcagta agaatacctc agaacatgga caactagatt tattataccc tagtattgac 1560 agtgagagtg aaatattacc aggggagaat caagtctttt atgataatag aactcaaaat 1620 gttgattatt tgaattctta ttattaccta gaatctcaaa aactaagtga taatgttgaa 1680 gattttactt ttacgagatc aattgaggag gctttggata atagtgcaaa agtatatact 1740 tactttccta cactagctaa taaagtaaat gcgggtgttc aaggtggttt atttttaatg 1800 tgggcaaatg atgtagttga agattttact acaaatattc taagaaaaga tacattagat 1860 aaaatatcag atgtatcagc tattattccc tatataggac ccgcattaaa tataagtaat 1920 tctgtaagaa gaggaaattt tactgaagca tttgcagtta ctggtgtaac tattttatta 1980 gaagcatttc ctgaatttac aatacctgca cttggtgcat ttgtgattta tagtaaggtt 2040 caagaaagaa acgagattat taaaactata gataattgtt tagaacaaag gattaagaga 2100 tggaaagatt catatgaatg gatgatggga acgtggttat ccaggattat tactcaattt 2160 aataatataa gttatcaaat gtatgattct ttaaattatc aggcaggtgc aatcaaagct 2220 aaaatagatt tagaatataa aaaatattca ggaagtgata aagaaaatat aaaaagtcaa 2280 gttgaaaatt taaaaaatag tttagatgta aaaatttcgg aagcaatgaa taatataaat 2340 aaatttatac gagaatgttc cgtaacatat ttatttaaaa atatgttacc taaagtaatt 2400 gatgaattaa atgagtttga tcgaaatact aaagcaaaat taattaatct tatagatagt 2460 cataatatta ttctagttgg tgaagtagat aaattaaaag caaaagtaaa taatagcttt 2520 caaaatacaa taccctttaa tattttttca tatactaata attctttatt aaaagatata 2580 attaatgaat atttcaataa tattaatgat tcaaaaattt tgagcctaca aaacagaaaa 2640 aatactttag tggatacatc aggatataat gcagaagtga gtgaagaagg cgatgttcag 2700 cttaatccaa tatttccatt tgactttaaa ttaggtagtt caggggagga tagaggtaaa 2760 gttatagtaa cccagaatga aaatattgta tataattcta tgtatgaaag ttttagcatt 2820 agtttttgga ttagaataaa taaatgggta agtaatttac ctggatatac tataattgat 2880 agtgttaaaa ataactcagg ttggagtata ggtattatta gtaatttttt agtatttact 2940 ttaaaacaaa atgaagatag tgaacaaagt ataaatttta gttatgatat atcaaataat 3000 gctcctggat agaataaatg gttttttgta actgttacta acaatatgat gggaaatatg 3060 aagatttata taaatggaaa attaatagat actataaaag ttaaagaact aactggaatt 3120 aattttagca aaactataac atttgaaata aataaaattc cagataccgg tttgattact 3180 tcagattctg ataacatcaa tatgtggata agagattttt atatatttgc taaagaatta 3240 gatggtaaag atattaatat attatttaat agcttgcaat atactaatgt tgtaaaagat 3300 tattggggaa atgatttaag atataataaa gaatattata tggttaatat agattattta 3360 aatagatata tgtatgcgaa ctcacgacaa attgttttta atacacgtag aaataataat 3420 gacttcaatg aaggatataa aattataata aaaagaatca gaggaaatac aaatgatact 3480 agagtacgag gaggagatat tttatatttt gatatgacaa ttaataacaa agcatataat 3540 ttgtttatga agaatgaaac tatgtatgca gataatcata gtactgaaga tatatatgct 3600 ataggtttaa gagaacaaac aaaggatata aatgataata ttatatttca aatacaacca 3660 atgaataata cttattatta ggcatctcaa atatttaaat caaattttaa tggagaaaat 3720 atttctggaa tatgttcaat aggtacttat cgttttagac ttggaggtga ttggtataga 3780 cacaattatt tggtgcctac tgtgaagcaa ggaaattatg cttcattatt agaatcaaca 3840 tcaactcatt ggggttttgt acctgtaagt gaataa 3876 5 3831 DNA botulinum toxin 5 atgacatggc cagtaaaaga ttttaattat agtgatcctg ttaatgacaa tgatatatta 60 tatttaagaa taccacaaaa taagttaatt actacacctg taaaagcttt tatgattact 120 caaaatattt gggtaatacc agaaagattt tcatcagata ctaatccaag tttaagtaaa 180 ccgcccagac ctacttcaaa gtatcaaagt tattatgatc ctagttattt atctactgat 240 gaacaaaaag atacattttt aaaagggatt ataaaattat ttaaaagaat taatgaaaga 300 gatataggaa aaaaattaat aaattattta gtagttggtt caccttttat gggagattca 360 agtacgcctg aagatacatt tgattttaca cgtcatacta ctaatattgc agttgaaaag 420 tttgaaaatg gtagttggaa agtaacaaat attataacac caagtgtatt gatatttgga 480 ccacttccta atatattaga ctatacagca tcccttacat tgcaaggaca acaatcaaat 540 ccatcatttg aagggtttgg aacattatct atactaaaag tagcacctga atttttgtta 600 acatttagtg atgtaacatc taatcaaagt tcagctgtat taggcaaatc tatattttgt 660 atggatccag taatagcttt aatgcatgag ttaacacatt ctttgcatca attatatgga 720 ataaatatac catctgataa aaggattcgt ccacaagtta gcgagggatt tttctctcaa 780 gatggaccca acgtacaatt tgaggaatta tatacatttg gaggattaga tgttgaaata 840 atacctcaaa ttgaaagatc acaattaaga gaaaaagcat taggtcacta taaagatata 900 gcgaaaagac ttaataatat taataaaact attccttcta gttggattag taatatagat 960 aaatataaaa aaatattttc tgaaaagtat aattttgata aagataatac aggaaatttt 1020 gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgt tatgtcagaa 1080 gttgtttatt cttcgcaata taatgttaaa aacaggactc attatttttc aaggcattat 1140 ctacctgtat ttgcaaatat attagatgat aatatttata ctataagaga tggttttaat 1200 ttaacaaata aaggttttaa tatagaaaat tcgggtcaga atatagaaag gaatcctgca 1260 ctacaaaagc ttagttcaga aagtgtagta gatttattta caaaagtatg tttaagatta 1320 acaaaaaata gtagagatga ttcaacatgt attaaagtta aaaataatag attaccttat 1380 gtagctgata aagatagcat ttcacaagaa atatttgaaa ataaaattat tacagatgag 1440 actaatgtac aaaattattc agataatttt tcattagatg aatctatttt agatgggcaa 1500 gttcctatta atcctgaaat agtagatcca ctattaccca atgttaatat ggaaccttta 1560 aatcttccag gtgaagaaat agtattttat gatgatatta ctaaatatgt tgattattta 1620 aattcttatt attatttgga atctcaaaaa ttaagtaata atgttgaaaa tattactctt 1680 acaacttcag ttgaagaagc attaggttat agcaataaga tatagacatt tttacctagc 1740 ttagctgaaa aagtgaataa aggtgttcaa gcaggtttat tcttaaattg ggcgaatgaa 1800 gtagttgagg attttactac aaatattatg aagaaagata cattggataa aatatcagat 1860 gtatcagtaa taattccata tataggacct gccttaaata taggaaattc agcattaagg 1920 ggaaatttta agcaagcatt tgcaacagct ggtgtagctt ttttattaga gggatttcca 1980 gagtttacta tacctgcact cggtgtattt accttttata gttctattca agaaagagag 2040 aaaattatta aaactataga aaattgtttg gaacaaagag ttaagagatg gaaagattca 2100 tatcaatgga tggtatcaaa ttggttgtca agaattacta ctcaatttaa tcatataaat 2160 tatcaaatgt atgattcttt aagttatcag gcagatgcaa tcaaagctaa aatagattta 2220 gaatataaaa aatagtcagg aagtgataaa gaaaatataa aaagtcaagt tgaaaattta 2280 aaaaatagtt tagatgtaaa aatttcggaa gcaatgaata atataaataa atttatacga 2340 gaatgttctg taacatagtt atttaaaaat atgctcccta aagtaattga cgaattaaat 2400 aagtttgatt taagaactaa aacagaatta attaatctta tagatagtca taatattatt 2460 ctagttggtg aagtagatag attaaaagca aaagtaaatg agagttttga aaatacaatg 2520 ccttttaata ttttttcata tactaataat tctttattaa aagatataat taatgaatat 2580 ttcaatagta ttaatgattc aaaaattttg agcttacaaa acaaaaaaaa tgctttagtg 2640 gatacatcag gatataatgc agaagtgagg gtaggagata atgttcaact taatacgata 2700 tatacaaatg actttaaatt aagtagttca ggagataaaa ttatagtaaa tttaaataat 2760 aatattttat atagcgctat ttatgagaac tctagtgtta gtttttggat taagatatct 2820 aaagatttaa ctaattctca taatgaatat acaataatta acagtataga acaaaattct 2880 gggtggaaat tatgtattag gaatggcaat atagaatgga ttttacaaga tgttaataga 2940 aagtataaaa gtttaatttt tgattatagt gaatcattaa gtcatacagg atatacaaat 3000 aaatggtttt ttgttactat aactaataat ataatggggt atatgaaact ttatataaat 3060 ggagaattaa agcagagtca aaaaattgaa gatttagatg aggttaagtt agataaaacc 3120 atagtatttg gaatagatga gaatatagat gagaatcaga tgctttggat tagagatttt 3180 aatatttttt ctaaagaatt aagtaatgaa gatattaata ttgtatatga gggacaaata 3240 ttaagaaatg ttattaaaga ttattgggga aatcctttga agtttgatac agaatattat 3300 attattaatg ataattatat agataggtat attgcacctg aaagtaatgt acttgtactt 3360 gttcggtatc cagatagatc taaattatat actggaaatc ctattactat taaatcagta 3420 tctgataaga atccttatag tagaatttta aatggagata atataattct tcatatgtta 3480 tataatagta ggaaatatat gataataaga gatactgata caatatatgc aacacaagga 3540 ggagagtgtt cacaaaattg tgtatatgca ttaaaattac agagtaattt aggtaattat 3600 ggtataggta tatttagtat aaaaaatatt gtatctaaaa ataaatattg tagtcaaatt 3660 ttctctagtt ttagggaaaa tacaatgctt ctagcagata tatataaacc ttggagattt 3720 tcttttaaaa atgcatagac gccagttgca gtaactaatt atgaaacaaa actattatca 3780 acttcatctt tttggaaatt tatttctagg gatccaggat gggtagagta a 3831 6 3753 DNA botulinum toxin 6 atgccaacaa ttaatagttt taattataat gatcctgtta ataatagaac aattttatat 60 attaaaccag gcggttgtca acaattttat aaatcattta atattatgaa aaatatttgg 120 ataattccag agagaaatgt aattggtaca attccccaag attttcttcc gcctacttca 180 ttgaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga tcaagaaaag 240 gataaatttt taaaaatagt cacaaaaata tttaatagaa taaatgataa tctttcagga 300 aggattttat tagaagaact gtcaaaagct aatccatatt taggaaatga taatactcca 360 gatggtgact tcattattaa tgatgcatca gcagttccaa ttcaattctc aaatggtagc 420 caaagcatac tattacctaa tgttattata atgggagcag agcctgattt atttgaaact 480 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacgg ttttggatca 540 atagctatag taacattctc acctgaatat tcttttagat ttaaagataa tagtatgaat 600 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga 660 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta 720 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta 780 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa 840 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaa tccttataaa 900 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaattta ttcggtaaat 960 ataaacaaat ttaatgatat ttttaaaaaa ttatacagct ttacggaatt tgatttagca 1020 actaaatttc aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt 1080 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat aaataattta 1140 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattac accaattaca 1200 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1260 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag 1320 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca 1380 aataataatt atgaaaatga tttagatcag gttattttaa attttaatag tgaatcagca 1440 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgctta tataccaaaa 1500 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaact taatgtattt 1560 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1620 attgatacag cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt 1680 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca acaagtatta 1740 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgc agatatttct 1800 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1860 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt 1920 ttaattccta caattttagt attcacgata aaatcttttt taggttcatc tgataataaa 1980 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa atggaaagaa 2040 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatt taataaaaga 2100 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg cacttaaagc aataatagaa 2160 tctaagtata atagttatac tttagaagaa aaaaatgagc ttacaaataa atatgatatt 2220 gagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg 2280 ttcttaactg aaagttctat atcttattta atgaaattaa taaatgaagt aaaaattaat 2340 aaattaagag aatatgatga aaatgttaaa acgtatttat tagattatat tataaaacat 2400 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaattga taccctaaat 2460 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt 2520 aataagttct ttaagagaat taaaagtagt tctgttttaa atatgagata taaaaatgat 2580 aaataggtag atacttcagg atatgattca aatataaata ttaatggaga tgtatataaa 2640 tatccaacta ataaaaatca atttggaata tataatgata aacttagtga agttaatata 2700 tctcaaaatg attacattat atatgataat aaatataaaa attttagtat tagtttttgg 2760 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaata cactataata 2820 aattgtatga gggataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt 2880 tggacattgc aagataattc aggaattaat caaaaattag catttaacta tggtaacgca 2940 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3000 ggagattcta aactttatat taatggaaat ttaatagata aaaaatcaat tttaaattta 3060 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga 3120 tatattggta ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa 3180 actttatata acaatgaacc taatgcaaat attttaaagg atttttgggg aaattatttg 3240 ctttatgaca aagaatagta tttattaaat gtgttaaaac caaataactt tattaatagg 3300 agaacagatt ctactttaag cattaataat ataagaagca ctattctttt agctaataga 3360 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat 3420 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttactt 3480 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat atcatcatct 3540 ggcaatagat ttaatcaagt agtagttatg aattcagtag gatgtacaat gaattttaaa 3600 aataataatg gaaataatat tgggttgtta ggtttcaagg cagatactgt agttgctagt 3660 acttggtatt atacacatat gagagataat acaaacagca atggattttt ttggaacttt 3720 atttctgaag aacatggatg gcaagaaaaa taa 3753 7 3759 DNA botulinum toxin 7 atgccaaaaa ttaatagttt taattataat gatcctgtta atgatagaac aattttatat 60 attaaaccag gcggttgtca agaattttat aaatcattta atattatgaa aaatatttgg 120 ataattccag agagaaatgt aattggtaca accccccaag attttcatcc gcctacttca 180 ttaaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga tgaagaaaag 240 gatagatttt taaaaatagt cacaaaaata tttaatagaa taaataataa tctttcagga 300 gggattttat tagaagaact gtcaaaagct aatccatatt tagggaatga taatactcca 360 gataatcaat tccatattgg tgatgcatca gcagttgaga ttaaattctc aaatggtagc 420 caagacatac tattacctaa tgttattata atgggagcag agcctgattt atttgaaact 480 aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacgg ttttggatca 540 atagctatag taacattctc acctgaatat tcttttagat ttaatgataa tagtatgaat 600 gaatttattc aagatcctgc tcttacatta atgcatgaat taatacattc attacatgga 660 ctatatgggg ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta 720 ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta 780 aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc tgattataaa 840 aaaatagcgt ctaaacttag caaagtacaa gtatctaatc cactacttaa tccttataaa 900 gatgtttttg aagcaaagta tggattagat aaagatgcta gcggaattta ttcggtaaat 960 ataaacaaat ttaatgatat ttttaaaaaa ttatagagct ttacggaatt tgatttagca 1020 actaaatttc aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt 1080 tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat aaataattta 1140 aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta gaattattac accaattaca 1200 ggtagaggac tagtaaaaaa aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1260 ataaggaaat caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag 1320 aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca 1380 aataataatt atgaaaatga tttagatcag gttattttaa attttaatag tgaatcagca 1440 cctggacttt cagatgaaaa attaaattta actatccaaa atgatgctta tataccaaaa 1500 tatgattcta atggaacaag tgatatagaa caacatgatg ttaatgaact taatgtattt 1560 ttctatttag atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1620 attgatacag cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt 1680 aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca acaagtgtta 1740 gtagatttta ctactgaagc taaccaaaaa agtactgttg ataaaattgc agatatttct 1800 atagttgttc catatatagg tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1860 tttaaagatg cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt 1920 ttaattccta caattttagt attcacgata aaatcttttt taggttcatc tgataataaa 1980 aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa atggaaagaa 2040 gtatatagtt ttatagtatc gaattggatg actaaaatta atacacaatt taataaaaga 2100 aaagaacaaa tgtatcaagc tttacaaaat caagtaaatg caattaaaac aataatagaa 2160 tctaagtata atagttatac tttagaggaa aaaaatgagc ttacaaataa atatgatatt 2220 aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg 2280 ttcttaactg aaagttctat atcctattta atgaaattaa taaatgaagt aaaaattaat 2340 aaattaagag aatatgatga gaatgtcaaa acgtatttat tgaattatat tatacaacat 2400 ggatcaatct tgggagagag tcagcaagaa ctaaattcta tggtaactga taccctaaat 2460 aatagtattc cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt 2520 aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagata taaaaatgat 2580 aaatacgtag atacttcagg atatgattca aatataaata ttaatggaga tgtatataaa 2640 tatccaacta ataaaaatca atttggaata tataatgata aacttagtga agttaatata 2700 tctcaaaatg attagattat atatgataat aaatataaaa attttagtat tagtttttgg 2760 gtaagaattc ctaactatga taataagata gtaaatgtta ataatgaata gactataata 2820 aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt 2880 tggacattgc aagataatgc aggaattaat caaaaattag catttaacta tggtaacgca 2940 aatggtattt ctgattatat aaataagtgg atttttgtaa ctataactaa tgatagatta 3000 ggagattcta aactttatat taatggaaat ttaatagatc aaaaatcaat tttaaattta 3060 ggtaatattc atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga 3120 tatattggta ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa 3180 actttatata gcaatgaacc taatacaaat attttgaagg atttttgggg aaattatttg 3240 ctttatgaca aagaatacta tttattaaat gtgttaaaac caaataactt tattgatagg 3300 agaaaagatt ctactttaag cattaataat ataagaagca ctattctttt agctaataga 3360 ttatatagtg gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat 3420 cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttattt 3480 ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat atcatcatct 3540 ggcaatagat ttaatcaagt agtagttatg aattcagtag gaaataattg tacaatgaat 3600 tttaaaaata ataatggaaa taatattggg ttgttaggtt tcaaggcaga tactgtagtt 3660 gctagtactt ggtattatac acatatgaga gatcatacaa acagcaatgg atgtttttgg 3720 aactttattt ctgaagaaca tggatggcaa gaaaaataa 3759 8 3825 DNA botulinum toxin 8 atgccagttg caataaatag ttttaattat aatgaccctg ttaatgatga tacaatttta 60 tagatgcaga taccatatga agaaaaaagt aaaaaatatt ataaagcttt tgagattatg 120 cgtaatgttt ggataattcc tgagagaaat acaataggaa cgaatcctag tgattttgat 180 ccaccggctt cattaaagaa cggaagcagt gcttattatg atcctaatta tttaaccact 240 gatgctgaaa aagatagata tttaaaaaca acgataaaat tatttaagag aattaatagt 300 aatcctgcag ggaaagtttt gttacaagaa atatcatatg ctaaaccata tttaggaaat 360 gaccacacgc caattgatga attctctcca gttactagaa ctacaagtgt taatataaaa 420 ttatcaacta atgttgaaag ttcaatgtta ttgaatcttc ttgtattggg agcaggacct 480 gatatatttg aaagttgttg ttaccccgtt agaaaactaa tagatccaga tgtagtttat 540 gatccaagta attatggttt tggatcaatt aatatcgtga cattttcacc tgagtatgaa 600 tatactttta atgatattag tggagggcat aatagtagta cagaatcatt tattgcagat 660 cctgcaattt cactagctca tgaattgata catgcactgc atggattata cggggctagg 720 ggagttactt atgaagagac tatagaagta aagcaagcac ctcttatgat agccgaaaaa 780 cccataaggc tagaagaatt tttaaccttt ggaggtcagg atttaaatat tattactagt 840 gctatgaagg aaaaaatata taacaatctt ttagctaact atgaaaaaat agctactaga 900 cttagtgaag ttaatagtgc tcctcctgaa tatgatatta atgaatataa agattatttt 960 caatggaagt atgggctaga taaaaatgct gatggaagtt atactgtaaa tgaaaataaa 1020 tttaatgaaa tttataaaaa attatatagt tttacagaga gtgacttagc aaataaattt 1080 aaagtaaaat gtagaaatac ttattttatt aaatatgaat ttttaaaagt tccaaatttg 1140 ttagatgatg atatttatac tgtatcagag gggtttaata taggtaattt agcagtaaac 1200 aatcgcggac aaagtataaa gttaaatcct aaaattattg attccattcc agataaaggt 1260 ctagtagaaa agatcgttaa attttgtaag agcgttattc ctagaaaagg tacaaaggcg 1320 ccaccgcgac tatgcattag agtaaataat agtgagttat tttttgtagc ttcagaaagt 1380 agctataatg aaaatgatat taatacacct aaagaaattg acgatacaac aaatctaaat 1440 aataattata gaaataattt agatgaagtt attttagatt ataatagtca gacaatacct 1500 caaatatcaa atcgaacatt aaatacactt gtacaagaca atagttatgt gccaagatat 1560 gattctaatg gaacaagtga aatagaggaa tatgatgttg ttgactttaa tgtatttttc 1620 tatttacatg cacaaaaagt gccagaaggt gaaaccaata taagtttaac ttcttcaatt 1680 gatacagcat tattagaaga atccaaagat atattttttt cttcagagtt tatcgatact 1740 atcaataaac ctgtaaatgc agcactattt atagattgga taagcaaagt aataagagat 1800 tttaccactg aagctacaca aaaaagtact gttgataaga ttgcagacat atctttaatt 1860 gtaccctatg taggtcttgc tttgaatata attattgagg cagaaaaagg aaattttgag 1920 gaggcatttg aattattagg agtgggtatt ttattagaat ttgtgccaga acttacaatt 1980 cctgtaattt tagtgtttac gataaaatcc tatatagatt catatgagaa taaaaataaa 2040 gcaattaaag caataaataa ttcattaatc gaaagagaag caaagtggaa agaaatatat 2100 agttggatag tatcaaattg gcttactaga attaatactc aatttaataa aagaaaagag 2160 caaatgtatc aggctttaca aaatcaagta gatgcaataa aaacagcaat agaatataaa 2220 tataataatt atacttcaga tgagaaaaat agacttgaat ctgaatataa tatcaataat 2280 atagaagaag aattgaataa aaaagtttct ttagcaatga aaaatataga aagatttatg 2340 acagaaagtt ctatatctta tttaatgaaa ttaataaatg aagccaaagt tggtaaatta 2400 aaaaaatatg ataaccatgt taagagcgat ttattaaact atattctcga ccatagatca 2460 atcttaggag agcagacaaa tgaattaagt gatttggtga ctagtacttt gaatagtagt 2520 attccatttg aactttcttc atatactaat gataaaattc taattatata ttttaataga 2580 ttatataaaa aaattaaaga tagttctatt ttagatatgc gatatgaaaa taataaattt 2640 atagatatct ctggatatgg ttcaaatata agcattaatg gaaacgtata tatttattca 2700 acaaatagaa atcaatttgg aatatataat agtaggctta gtgaagttaa tatagctcaa 2760 aataatgata ttatatagaa tagtagatat caaaatttta gtattagttt ctgggtaagg 2820 attcctaaac actagaaacc tatgaatcat aatcgggaat agactataat aaattgtatg 2880 gggaataata attcgggatg gaaaatatca cttagaactg ttagagattg tgaaataatt 2940 tggactttac aagatacttc tggaaataag gaaaatttaa tttttaggta tgaagaactt 3000 aataggatat ctaattatat aaataaatgg atttttgtaa ctattactaa taatagatta 3060 ggcaattcta gaatttagat caatggaaat ttaatagttg aaaaatcaat ttcgaattta 3120 ggtgatattc atgttagtga taatatatta tttaaaattg ttggttgtga tgatgaaacg 3180 tatgttggta taagatattt taaagttttt aatacggaat tagataaaac agaaattgag 3240 actttatata gtaatgagcc agatccaagt atcttaaaaa actattgggg aaattatttg 3300 ctatataata aaaaatatta tttattcaat ttactaagaa aagataagta tattactctg 3360 aattcaggca ttttaaatat taatcaacaa agaggtgtta ctgaaggctc tgtttttttg 3420 aactataaat tatatgaagg agtagaagtc attataagaa aaaatggtcc tatagatata 3480 tctaatacag ataattttgt tagaaaaaac gatctagcat acattaatgt agtagatcgt 3540 ggtgtagaat atcggttata tgctgataca aaatcagaga aagagaaaat aataagaaca 3600 tctaatctaa acgatagctt aggtcaaatt atagttatgg attcaatagg aaataattgc 3660 acaatgaatt ttcaaaacaa taatgggagc aatataggat tactaggttt tcattcaaat 3720 aatttggttg ctagtagttg gtattataac aatatacgaa gaaatactag cagtaatgga 3780 tgcttttgga gttctatttc taaagagaat ggatggaaag aatga 3825 9 3894 DNA Artificial Sequence Description of Artificial Sequence synthetic primers used to introduce Stu I and EcoR I restriction sites into the 5′ and 3′ ends of the BoNT/A-L chain gene fragment 9 atgccagtta atataaaaaa ctttaattat aatgacccta ttaataatga tgacattatt 60 atgatggaac cattcaatga cccagggcca ggaacatatt ataaagcttt taggattata 120 gatcgtattt ggatagtacc agaaaggttt acttatggat ttcaacctga ccaatttaat 180 gccagtacag gagtttttag taaagatgtc tacgaatatt aggatccaac ttatttaaaa 240 accgatgctg aaaaagataa atttttaaaa acaatgatta aattatttaa tagaattaat 300 tcaaaaccat caggacagag attactggat atgatagtag atgctatacc ttatcttgga 360 aatgcatcta caccgcccga caaatttgca gcaaatgttg caaatgtatc tattaataaa 420 aaaattatcc aacctggagc tgaagatcaa ataaaaggtt taatgacaaa tttaataata 480 tttggaccag gaccagttct aagtgataat tttactgata gtatgattat gaatggccat 540 tccccaatat cagaaggatt tggtgcaaga atgatgataa gattttgtcc tagttgttta 600 aatgtattta ataatgttca ggaaaataaa gatacatcta tatttagtag acgcgcgtat 660 tttgcagatc cagctctaac gttaatgcat gaacttatac atgtgttaca tggattatat 720 ggaattaaga taagtaattt accaattact ccaaatacaa aagaattttt catgcaacat 780 agcgatcctg tacaagcaga agaactatat acattcggag gacatgatcc tagtgttata 840 agtccttcta cggatatgaa tatttataat aaagcgttac aaaattttca agatatagct 900 aataggctta atattgtttc aagtgcccaa gggagtggaa ttgatatttc cttatataaa 960 caaatatata aaaataaata tgattttgtt gaagatccta atggaaaata tagtgtagat 1020 aaggataagt ttgataaatt atataaggcc ttaatgtttg gctttactga aactaatcta 1080 gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatattt gccaccgata 1140 aaaactgaaa aattgttaga caatacaatt tatactcaaa atgaaggctt taacatagct 1200 agtaaaaatc tcaaaacgga atttaatggt cagaataagg cggtaaataa agaggcttat 1260 gaagaaatca gcctagaaca tctcgttata tatagaatag caatgtgcaa gcctgtaatg 1320 tacaaaaata ccggtaaatc tgaacagtgt attattgtta ataatgagga tttatttttc 1380 atagctaata aagatagttt ttcaaaagat ttagctaaag cagaaactat agcatataat 1440 acacaaaata atactataga aaataatttt tctatagatc agttgatttt agataatgat 1500 ttaagcagtg gcatagactt accaaatgaa aacacagaac catttacaaa ttttgacgac 1560 atagatatcc ctgtgtatat taaacaatct gctttaaaaa aaatttttgt ggatggagat 1620 agcctttttg aatatttaca tgctcaaaca tttccttcta atatagaaaa tctacaacta 1680 acgaattcat taaatgatgc tttaagaaat aataataaag tctatacttt tttttctaca 1740 aaccttgttg aaaaagctaa tacagttgta ggtgcttcac tttttgtaaa ctgggtaaaa 1800 ggagtaatag atgattttac atctgaatcc acacaaaaaa gtactataga taaagtttca 1860 gatgtatcca taattattcc ctatatagga cctgctttga atgtaggaaa tgaaacagct 1920 aaagaaaatt ttaaaaatgc ttttgaaata ggtggagccg ctatcttaat ggagtttatt 1980 ccagaactta ttgtacctat agttggattt tttacattag aatcatatgt aggaaataaa 2040 gggcatatta ttatgacgat atccaatgct ttaaagaaaa gggatcaaaa atggacagat 2100 atgtatggtt tgatagtatc gcagtggctc tcaacggtta atactcaatt ttatacaata 2160 aaagaaagaa tgtagaatgc tttaaataat caatcacaag caatagaaaa aataatagaa 2220 gatcaatata atagatatag tgaagaagat aaaatgaata ttaacattga ttttaatgat 2280 atagatttta aacttaatca aagtataaat ttagcaataa acaatataga tgattttata 2340 aaccaatgtt ctatatcata tctaatgaat agaatgattc cattagctgt aaaaaagtta 2400 aaagactttg atgataatct taagagagat ttattggagt atatagatac aaatgaacta 2460 tatttacttg atgaagtaaa tattctaaaa tcaaaagtaa atagacacct aaaagacagt 2520 ataccatttg atctttcact atataccaag gacacaattt taatacaagt ttttaataat 2580 tatattagta atattagtag taatgctatt ttaagtttaa gttatagagg tgggcgttta 2640 atagattcat ctggatatgg tgcaactatg aatgtaggtt cagatgttat ctttaatgat 2700 ataggaaatg gtcaatttaa attaaataat tctgaaaata gtaatattac ggcacatcaa 2760 agtaaattcg ttgtatatga tagtatgttt gataatttta gcattaactt ttgggtaagg 2820 actcctaaat ataataataa tgatatacaa acttatcttc aaaatgagta tacaataatt 2880 agttgtataa aaaatgactc aggatggaaa gtatctatta agggaaatag aataatatgg 2940 acattaatag atgttaatgc aaaatctaaa tcaatatttt tcgaatatag tataaaagat 3000 aatatatcag attatataaa taaatggttt tccataacta ttactaatga tagattaggt 3060 aacgcaaata tttatataaa tggaagtttg aaaaaaagtg aaaaaatttt aaacttagat 3120 agaattaatt ctagtaatga tatagacttc aaattaatta attgtacaga tactactaaa 3180 tttgtttgga ttaaggattt taatattttt ggtagagaat taaatgctac agaagtatct 3240 tcactatatt ggattcaatc atctacaaat actttaaaag atttttgggg gaatccttta 3300 agataggata cacaatacta tctgtttaat caaggtatgc aaaatatcta tataaagtat 3360 tttagtaaag cttctatggg ggaaactgca ccacgtacaa actttaataa tgcagcaata 3420 aattatcaaa atttatatct tggtttacga tttattataa aaaaagcatc aaattctcgg 3480 aatataaata atgataatat agtcagagaa ggagattata tatatcttaa tattgataat 3540 atttctgatg aatcttagag agtatatgtt ttggtgaatt ctaaagaaat tcaaactcaa 3600 ttatttttag cacccataaa tgatgatcct acgttctatg atgtactaca aataaaaaaa 3660 tattatgaaa aaacaacata taattgtcag atactttgcg aaaaagatac taaaacattt 3720 gggctgtttg gaattggtaa atttgttaaa gattatggat atgtttggga tacctatgat 3780 aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatat aaataaatta 3840 aggttgggat gtaattggca attcattccc gtggatgaag gatggacaga ataa 3894 10 23 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide used to introduce Stu I and EcoR I into BoNT/A-L chain gene fragments 10 aaaggccttt tgttaataaa caa 23 11 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide used to introduce Stu I and EcoR I into BoNT/A-L chain gene fragment 11 ggaattctta cttattgtat ccttta 26 12 13 PRT Artificial Sequence Description of Artificial Sequence polypeptide fragment used to raise antibodies 12 Cys Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 1 5 10

Claims

What is claimed is:

1. A method for treating hyperhidrosis in a mammal, said method comprising the step of locally administering a drug particle to an affected skin area without using a needle.

2. The method of claim 1 wherein the skin comprises an epidermis layer, a dermis layer and a hypodermis layer.

3. The method of claim 1 wherein the drug particle is administered to a dermis layer of the skin.

4. The method of claim 1 wherein the drug particle is administered to one or more layers of the skin where a sweat gland and/or a nerve innervating a sweat gland is located.

5. The method of claim 1 wherein the drug particle is administered to the skin and substantially not to a muscle tissue.

6. The method of claim 1 wherein the drug particle is administered to a dermis layer of the skin and substantially not to a muscle tissue.

7. The method of claim 1 wherein the step of administering includes using a needleless injector.

8. The method of claim 7 wherein the drug particle is administered to a dermis layer of a skin and substantially not to a muscle tissue.

9. The method of claim 1 wherein the drug particle comprises a neurotoxin.

10. The method of claim 9 wherein the neurotoxin comprises:

(a) a targeting component;

(b) a therapeutic component; and

(c) a translocation component.

11. The method of claim 10 wherein the targeting component binds to a presynaptic nerve terminal.

12. The method of claim 12 wherein the presynaptic nerve terminal belongs to a cholinergic neuron.

13. The method of claim 11 wherein the targeting component comprises a carboxyl end segment of a heavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E, F, G or a variant thereof.

14. The method of claim 11 wherein the targeting component comprises a carboxyl end fragment of a heavy chain of a botulinum toxin type A.

15. The method of claim 11 wherein the therapeutic component substantially interferes with the release of neurotransmitters from a neuron or its terminals.

16. The method of claim 11 wherein the therapeutic component comprises a light chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E, F, G or a variant thereof.

17. The method of claim 11 wherein the therapeutic component comprises a light chain of a botulinum toxin type A.

18. The method of claim 11 wherein the translocation component facilitates the transfer of at least a part of the neurotoxin into the cytoplasm of the target cell.

19. The method of claim 11 wherein the translocation component comprises an amino end fragment of a heavy chain of a butyricum toxin, a tetani toxin, a botulimum toxin type A, B, C₁, D, E, F, G or a variant thereof.

20. The method of claim 11 wherein the translocation component comprises an amino end fragment of a heavy chain of a botulinum toxin type A.

21. The method of claim 11 wherein the targeting component comprises a carboxyl end fragment of a heavy chain of a botulinum toxin type A, the therapeutic component comprises a light chain of a botulinum toxin type A and the translocation component comprises an amine end fragment of a heavy chain of a botulinum toxin type A.

22. The method of claim 11 wherein the neurotoxin is botulinum toxin type A.

23. The method of claim 1 wherein the neurotoxin is recombinantly produced.

24. The method of claim 1 wherein the drug particle comprises a neurotoxin and a carrier, wherein the neurotoxin is coated onto the carrier.

25. The method of claim 24 wherein the carrier is a dense material selected from the group consisting of gold, platinum and ice crystal.

26. The method of claim 9 wherein the neurotoxin comprises a nucleotide sequence.

27. The method of claim 26 wherein the nucleotide sequence is SEQ. ID. #1, variants thereof or fragments thereof.

28. The method of claim 26 wherein the nucleotide sequence is selected from the group consisting of SEQ. ID #2, SEQ. ID. #3, SEQ. ID. #4, SEQ. ID. #5, SEQ. ID. #6, SEQ. ID. #7, SEQ. ID. #8 and SEQ. ID. #9, variants thereof or fragments thereof.

29. A method for treating hyperhidrosis in a mammal, said method comprising the step of using a needleless injector to locally administer a drug particle comprising a neurotoxin to a dermis layer of an affected area of a skin.

30. The method of claim 29 wherein the neurotoxin comprises botulinum toxin type A.