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Title:  Proteins within the type E botulinum neurotoxin complex

United States Patent:  6,699,966

Issued:  March 2, 2004

Inventors:  Singh; Bal Ram (Dartmouth, MA); Zhang; Zhong (New Bedford, MA)

Assignee:  University of Massachusetts (Boston, MA)

Appl. No.:  546136

Filed:  April 10, 2000

Abstract

The invention features a polypeptide complex synthesized by bacteria of the genus Clostridia that contains the serotype E botulinum neurotoxin and five neurotoxin associated polypeptides having molecular weights of about 118, 80, 65, 40, and 18 kDa, respectively. The complex is useful in the treatment of diseases or conditions that are caused by excessive release of acetylcholine from presynaptic nerve terminals.

SUMMARY OF THE INVENTION

The invention is based on the discovery that the type E botulinum toxin exists in a complex that includes the toxin and five other polypeptides termed neurotoxin associated proteins (NAPs). This discovery is contrary to the prior assertions of those in the field, who believed that the toxin was associated with only one other polypeptide, a neurotoxin binding protein (NBP) having a molecular weight of approximately 118 kDa.

Accordingly, the invention features a substantially pure polypeptide complex that includes a Clostridium botulinum neurotoxin and more than one Closiridium botulinum type E neurotoxin associated polypeptide. The polypeptides of the invention include the newly discovered NAPs, which have molecular weights of approximately 80, 65, 40, and 18 kDa, and substantially pure antibodies that specifically bind to one or more of these polypeptides. or complexes of one or more of the NAPs with type E botulinum toxin, or toxins from other Clostridium botulinum serotypes including A, B, C, D, F, and G.

The following peptide sequences have been obtained: (1) MKQAFVFEFD (SEQ ID NO:1), from the 18 kDa polypeptide; (2) MRINTNINSM (SEQ ID NO:2), from the 40 kDa polypeptide; (3) MQTTTLNWDT (SEQ ID NO:3), from the 65 kDa polypeptide; and (4) TNLKPYIIYD (SEQ ID NO:4), from the 80 kDa polypeptide. In addition, the complete amino acid sequence of the 18 kDa polypeptide has been obtained and is shown in FIG. 8 (SEQ ID NO:5).

The compositions of the invention (e.g., the novel NAPs and antibodies that specifically bind to them) can be used to detect the serotype E neurotoxin complex in a sample. For example, one can contact the sample with an antibody that specifically binds a NAP, or with a NAP that binds the neurotoxin itself (as described in the Examples, below, the NAP having an approximate molecular weight of 80 kDa binds directly to the type E neurotoxin) and detect, if present, antibody-bound type E associated polypeptide or NAP-bound type E neurotoxin. The presence of these antibody complexes indicates the presence of serotype E neurotoxin in the sample. The detection methods of the invention can be used to examine virtually any type of sample, including samples of foodstuffs, or biological samples, such as gastrointestinal, blood, or tissue samples obtained from a vertebrate animal.

The novel compositions of the invention also provide the basis for methods of treating patients who suffer from a disease or conditions associated with excessive release of acetylcholine from presynaptic nerve terminals. The patient is treated by administration of a therapeutically effective amount of a polypeptide complex that contains the serotype E neurotoxin (or other serotype toxin) and more than one NAP, e.g., one or more of the 80, 65, 40, and 18 kDa NAPs, and/or the 118 kDa polypeptide. The conditions associated with excessive acetylcholine release include undesirable contractions of smooth or skeletal muscle cells, which can, in turn, cause spasmodic torticollis, essential tremor, spasmodic dysphonia, charley horse, strabismus, blepharospasm, oromandibular dystonia, spasms of the sphincters of the cardiovascular, gastrointestinal, or urinary systems, or tardive dyskinesia. Excessive release of acetylcholine can also cause profuse sweating, lacrimation, or mucous secretion. Patients who could benefit from the methods described herein include those who suffer from spasticity that occurs secondary to another event such as brain ischemia, traumatic injury of the brain or spinal cord, tension headache, or pain (e.g., pain caused by sporting injuries or arthritis).

In addition, the novel compositions of the invention can be formulated as a vaccine and administered to an animal in an amount sufficient to confer a degree of protection against serotype E (or other) neurotoxin.

Administration of a purified neurotoxin complex (e.g., of the type E (or other) neurotoxin and more than one of the new NAPs), as described below, is superior to administration of the neurotoxin alone because, within the complex, the neurotoxin is more stable and thus, longer-lasting. This feature minimizes the frequency of administration and thereby reduces any risk, discomfort, or inconvenience that the patient may experience.

The type E complex is a superior therapeutic agent, relative to the other botulinum serotypes, because the activity of the type E neurotoxin can be enhanced 100-fold by treatment with trypsin, which breaks the bonds between the two polypeptide chains that constitute the neurotoxin. Therefore, application of the type E neurotoxin complex can be controlled by trypsinization, in a way that allows graded release of the neurotoxin from the complex. This unique mechanism provides more controlled and longer-lasting effects than would otherwise be possible.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

Contrary to the general understanding in the field, the type E botulinum toxin complex consists in vivo of the neurotoxin, which has a molecular weight of about 150 kDa, and five (not one, as previously believed) polypeptides that form a complex with the neurotoxin. These five polypeptides have molecular weights of approximately 118, 80, 65, 40, and 18 kDa. Those of skill in the art will recognize that the measured or apparent molecular weight of a polypeptide can vary depending, for example, on the number of glycosylated residues, and even on the method used to determine the molecular weight. Accordingly, the measured molecular weights of the NAPs of the invention can vary. For example, the measured molecular weight of the approximately 80 kDa polypeptide can vary between 70 and 90 kDa; the measured molecular weight of the approximately 65 kDa polypeptide can vary between 60 and 70 kDa; the measured molecular weight of the approximately 40 kDa polypeptide can vary between 35 and 45 kDa; and the measured molecular weight of the approximately 18 kDa polypeptide can vary between 15 and 21 kDa.

The novel polypeptides discovered in the type E botulinum neurotoxin complex can be used in a number of ways. First, they can be used to detect the presence of the type E botulin toxin in a sample, such as a food sample or a sample of biological tissue, or to generate antibodies that can be used in analogous detection methods. For example, if a patient is exposed to the neurotoxin, the NAPs and NAP-binding antibodies of the invention provide the means (through direct binding detection systems or antibody-based detection systems) for rapid and reliable diagnosis. The NAPs, in their naturally occurring complex with the type E neurotoxin (or complexed individually or in groups with type E or other neurotoxins), are also useful in treating diseases associated with excessive release of acetylcholine from cholinergic nerve terminals and, in addition, they can be used to generate vaccines for immunization. These uses are described in further detail below.

Polypeptides of the Invention

The polypeptides of the invention include NAPs having molecular weights of approximately 80, 65, 40, and 18 kDa (alone or in any combination, particularly in a combination that includes the type E (or other) botulin neurotoxin, and/or the neurotoxin binding protein (NBP) having a molecular weight of approximately 118 kDa), and antibodies that specifically bind to one or more of these NAPs or NAP complexes.

The invention encompasses fulllength NAPs as well as fragments that correspond to functional domains of the NAPs (e.g., fragments that bind to polypeptides in the type E botulin complex and help to increase the stability of the neurotoxin in vitro or in vivo, or fragments that are antigenic (i.e., that elicit the production of antibodies)). The NAPs of the invention, and fragments thereof, can have the sequence of a wild-type NAP, or can contain a mutation (including deletions, additions, or substitutions of one or more amino acid residues). Preferably, the mutant polypeptides retain at least 50%, 75%, or at least 95% or more of the biological activity of the corresponding wild-type polypeptide. It is a straightforward matter to compare the biological activities of the mutant and wild-type polypeptides. They can, for example, be used in side-by-side tests for lethality in rodents. If an equivalent number of animals are killed regardless of whether they receive a particular dose of a type E complex containing wild-type or mutant NAPs, the mutant NAPs would be said to retain the biological activity of the wild-type NAPs. If only half as many animals die following administration of a complex containing a mutant NAP, then the mutant would be said to retain half the biological activity of the wild-type NAP. Those of skill in the art will be aware of numerous ways in which biological activities can be fairly compared.

Mutant NAPs that contain a substitution of one or more amino acid residues can be made purposefully or randomly (e.g., by using routine techniques of recombinant DNA engineering or random mutagenesis, respectively). Amino acid substitutions may be purposefully changed to affect the polarity, charge, solubility, hydrophobicity, hydrophilicity, or amphipathic nature of the residues involved. Not only may the mutant polypeptides retain biological activity, this activity could be increased. For example, a mutant NAP could be made to bind with a higher affinity to the type E botulin neurotoxin.

Polypeptides in which the NAPs are fused to an unrelated protein (e.g., a protein that can be easily detected or quantitated) are also considered within the scope of the invention.

The polypeptides of the invention can be purified from a naturallyoccurring source, chemically synthesized (e.g., see Creighton, In Proteins: Structures and Molecular Principles, W.H. Freeman & Co., New York, N.Y., 1983), or produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acids. These methods can, for example, be used to construct expression vectors containing a NAP encoding sequence, and appropriate transcriptional and translational control signals.

"Substantially pure" polypeptides are polypeptides in preparations in which they represent at least 60% by weight of the preparation. When one or more NAPs are present in a complex, for example, in a complex with the type E botulin neurotoxin, a substantially pure preparation will consist of at least 60%, by weight, the polypeptides of the given complex. Preferably, preparations containing one or more NAPs are at least 75%, at least 90%, or even at least 99%, by weight, the polypeptide(s) of interest. Purity can be readily measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

The amino acid sequence of the NAP having a molecular weight of approximately 18 kDa has been determined (FIG. 8; SEQ ID NO:5), and partial sequences are described herein for the NAPs having molecular weights of approximately 80 (SEQ ID NO:4), 65 (SEQ ID NO:3), and 40 kDa (SEQ ID NO:2). It is well within the abilities of those of ordinary skill in the art to obtain additional sequence information from the partial sequences disclosed herein. For example, PCR technology can be used to isolate full length NAP cDNA sequences as follows. First, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source (e.g., a bacterium of the genus Clostridia), and reverse transcribed using an oligonucleotide primer specific for the most 5' end of the amplified fragment. This oligonucleotide primes first strand synthesis. The resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal transferase reaction, and the hybrid can be digested with RNAse H. Second strand synthesis can then be primed with a poly-C primer. The sequence of the amplified fragment can then be obtained using any number of routine procedures.

Production of Antibodies Against the Type E Neurotoxin Associated Proteins

Antibodies that specifically bind to one or more epitopes of a NAP, or fragments or derivatives thereof, are also considered within the scope of the present invention. An antibody is said to "specifically bind" to a polypeptide when it recognizes and binds to that polypeptide, but not to other molecules in a sample (e.g., a biological sample that includes type E neurotoxin associated polypeptides). The antibodies of the invention can be polyclonal, monoclonal, humanized, chimeric, or single chain antibodies, or Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, antiidiotype (anti-Id) antibodies, and epitopebinding fragments of any of the types of antibodies listed above.

A variety of standard methods can be used to generate antibodies against the type E neurotoxin associated proteins. For example, the type E neurotoxin associated proteins, either individually or in their complexed forms, can be administered to an animal, such as a rat, mouse, or rabbit, to induce the production of polyclonal antibodies. Alternatively, antigenic fragments of the individual polypeptides can be used to generate polyclonal antibodies. Various adjuvants can be used to increase the immunological response to an antigen, depending on the host species. These adjuvants include Freund's (complete or incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentially useful human adjuvants are known to include BCG (bacile CalmetteGuerin) and Corynebacterium parvum.

In addition, antibodies according to the invention can be monoclonal antibodies (i.e., antibodies from a homogenous population that recognize a particular antigen) that are generated by using either individual serotype E NAPs, the intact type E complex, or complexes of the neurotoxin with the NBP and any one or more of the new NAPs. Such monoclonal antibodies can be prepared using standard hybridoma technology (see; e.g., Kohler et al., Nature256:495, 1975; Kohler et al., Eur. J. Immunol. 6:292, and 6:511, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981; Kruisbeck et al., Hornbeck et al., and Yokoyama, In Current Protocols in Immunology, Vol. 1, New York, John Wiley & Sons, Inc., 1994). Monoclonal antibodies can be of any immunoglobulin class, including the IgG, IgM, IgE, IgA, and IgD classes, and any subclass thereof. The hybridoma producing the mAb of the invention can be cultivated in vitro or in vivo.

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-7955, 1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature 314:452-454, 1985) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA-85:5879-5883, 1988; and Ward et al., Nature 334:544-546, 1989) can be adapted to produce single chain antibodies against NAPs. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes can also be generated by known techniques. For example, such fragments include, but are not limited to: the F(ab')2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science 246:1275-1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

The binding specificity and activity of purified antitype E (or other serotypes) complex antibodies, such as those described above, can be confirmed by testing their ability to interfere with the biological activity of the neurotoxin and/or the complex. This ability can be tested by adding the antibodies to any number of standard in vitro assays in which the release of acetylcholine from presynaptic nerve terminals can be monitored. These assays include preparations of different neuromuscular junctions, such as the mouse phrenic nerve hemidiaphragm, the mouse plantar nervelumbrical muscle, and chick ciliary ganglioniris muscle preparations (Bandyopadhyay et al., J. Biol. Chem. 262:2660-2663, 1987); Bittner et al., J. Biol. Chem. 264:10354-10360, 1989; Clark et al., J. Neurosci. Methods 19:285-295, 1987; and Lomneth et al., Neurosci. Lett., 113:211-216, 1990). The binding specificity and activity of any given antibody is tested by determining whether that antibody effectively blocks the action of type E neurotoxin complex applied at the neuromuscular junction.

Polypeptide-Based Detection Systems for Type E Neurotoxin Associated Proteins

The NAPs described herein have a variety of uses, including the detection of type E neurotoxin. The type A neurotoxin remains associated with its protein complex both in bacterial culture medium and in natural cases of food poisoning (Sakaguchi, Pharmac. Ther. 19:165-194, 1983). Given this evidence, it is likely that the 118 kDa binding protein and the other four, lower molecular weight members of the type E complex also remain associated with the cognate toxin in vitro and in vivo. In addition, neurotoxin associated proteins have been shown to be more immunogenic than the neurotoxin itself (Singh et al., Toxicon 34:267-275, 1996).

Antibodies generated against any one of the NAPs or combinations thereof can also be used to detect the type E neurotoxin complex using various standard methods. For example, the antibodies can be used with a fiber opticbased biosensor, as described herein, which uses an evanescent wave from a tapered optical fiber for signal discrimination. This antibody-based "sandwich" immunoassay detection system can detect botulinum toxin much more quickly than any method currently available, but other immunoassay methods can be used. The actual signal collection period with the biosensor is less than one minute. Detection is accomplished using a twostep sandwich immunoassay. Antibodybound optical fibers are incubated in a solution of type E complex, and a signal is generated when the fiber bound complex binds a fluorescently labeled antibody (see, Ogert et al., Anal. Biochem. 205:306-312, 1992; and Singh et al., In Natural Toxins 11, B. R. Singh and A. Tu, Eds., Plenum Press, pp. 498-508, 1996).

One of the problems historically associated with sandwich immunoassays is that the first antibody (here, the antibody bound to the optical fiber) and the second antibody (here, the antibody added to detect the fiberbound complex), compete for the same epitope on the neurotoxin. To circumvent this problem, two antibodies can be used. The first specifically binds to one portion of the neurotoxin or one NAP of the type E complex, which will be attached to the fiber, and a second specifically binds to either a second portion of the neurotoxin or a second member of the polypeptide complex, which would specifically recognize the fiberbound complex.

Any polypeptide (be it a NAP or an antibody of the invention) can be detectably labeled to facilitate the detection of the type E botulin complex. For example, the polypeptides can be conjugated with a radioopaque or other appropriate compound, such as a fluorescent compound, that can be brought into contact with a sample that may contain the botulin complex. Alternatively, the polypeptide can be linked to an enzyme and used in an enzyme immunoassay (EIA; Voller et al. J. Clin. Pathol. 31:507-520, 1978; Butler, Methods Enzymol. 73:482-523, 1981). The enzyme that is bound to the antibody will react with an appropriate substrate, e.g., a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Enzymes that can be used to detectably label a polypeptide include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, betagalactosidase, alkaline phosphatase, ribonuclease, and urease.

Preparation and Administration of A Neurotoxin Vaccine

The invention also includes a vaccine composition containing a type E (or other serotype) neurotoxin and more than one type E neurotoxin associated polypeptide (or immunogenic fragment or derivative thereof) and a pharmaceutically acceptable diluent or carrier, such as phosphate buffered saline or a bicarbonate solution (e.g., 0.24 M NaHCO3). The carriers and diluents used in the invention are selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. An adjuvant, for example, a cholera toxin, Escherichia coli heat labile enterotoxin (LT), or a fragment or derivative thereof having adjuvant activity, may also be included in the vaccine composition of the invention.

Skilled artisans can obtain further guidance in the preparation of a vaccine for type E neurotoxin complex in Singh et al. (Toxicon 27:403-410, 1990). Briefly, approximately 1.5 mg of the type E complex is added to approximately 10 ml of 0.05 M sodium citrate buffer (pH 5.5) and dialyzed against 0.39% formaldehyde at 30oC. for 7 days. The formaldehyde-containing buffer is replaced every day with fresh buffer solution. The detoxified neurotoxin (toxoid or vaccine) is dialyzed against 0.1 M sodium phosphate buffer (pH 7.4) without formaldehyde for two days with several changes of buffer.

The amount of vaccine administered will depend, for example, on the particular vaccine antigen, whether an adjuvant is co-administered with the antigen, the type of adjuvant co-administered, the mode and frequency of administration, and the desircd effect (e.g., protection or treatment), as can be determined by one skilled in the art. In general, the vaccine antigens of the invention are administered in amounts ranging between, for example, 1 .mu.g and 100 mg. If adjuvants are administered with the vaccines, amounts of the polypeptide vaccine ranging between, for example, 1 ng and 1 mg can be used. Administration is repeated as necessary, as can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly intervals.

Treatment with Polypeptides in the Type E Neurotoxin Complex

Any disease or discomfort associated with an exaggerated release of acetylcholine from a presynaptic nerve terminal can be treated with the purified or isolated type E botulinum neurotoxin complex described herein, or with complexes formed of other serotype toxins combined with one or more of the NAPS described herein. Those of skill in the art are aware of the normal parameters for acetylcholine release and of the normal range of physiological function that is produced when an appropriate amount of this neurotransmitter is released onto a muscle from adjacent nerve terminals. An exaggerated release of acetylcholine would be any level of release that exceeds the normal parameters and causes aberrant physiological function. The diseases or conditions associated with an exaggerated release of acetylcholine can involve spasms of either smooth or skeletal muscle cells. More specifically, these diseases or conditions include spasmodic torticollis, essential tremor, spasmodic dysphonia, charley horse, strabismus, blepharospasm, oromandibular dystonia, spasms of the sphincters of the cardiovascular, gastrointestinal, or urinary systems, and tardive dyskinesia, which may result from treatment with an anti-psychotic drug such as THORAZINE.RTM. or HALDOL.RTM..

For example, an adult male patient suffering from tardive dyskinesia resulting from treatment with an anti-psychotic drug can be treated with 50-200 units (defined below) of Botulinum type E complex by direct injection into the facial muscles. Within three days, the symptoms of tardive dyskinesia, i.e., orofacial dyskinesia, athetosis, dystonia, chorea, tics and facial grimacing are markedly reduced.

Spasticity that occurs secondary to brain ischemia, or traumatic injury of the brain or spinal cord, are similarly amenable to treatment.

In instances where the postsynaptic target is a gland, nerve plexus, or ganglion, rather than a muscle, the type E complex can be administered to control profuse sweating, lacrimation, and mucous secretion. For example, an adult male patient with excessive unilateral sweating can be treated by administering 0.01 to 50 units of type E botulinum complex to the gland nerve plexus, ganglion, spinal cord, or central nervous system. Preferably, the nerve plexus or ganglion that malfunctions to produce the excessive sweating is treated directly. Administration of type E neurotoxin complex to the spinal cord or brain, while feasible, may cause general weakness.

Other conditions that can be treated include tension headache and pain caused by sporting injuries or arthritic contractions. If necessary, overactive muscles can be identified with electromyography (EMG).

While it is expected that the methods of the invention will most commonly be applied to human patients, domestic pets (such as dogs and cats) and livestock (such as cows, sheep, and pigs) can also be treated with the compositions described herein.

Administration of Polypeptides within the Type E Neurotoxin Complex

The dose of type E neurotoxin complex administered to a patient will depend generally upon the severity of the condition, the age, weight, sex, and general health of the patient, and the potency of the toxin, which is expressed as a multiple of the LD50 value for the mouse.

The dosages used in human therapeutic applications are roughly proportional to the mass of muscle to be treated. Typically, the dose administered to the patient is from about 0.01 to about 1,000 units, for example, about 500 units. A unit is defined as the amount of type E neurotoxin (or type E neurotoxin complex) that kills 50% of a group of mice (typically a group of 18-20 female mice that weigh on average 20 grams). The dosage is adjusted, either in quantity or frequency, to achieve sufficient reduction in acetylcholine release to afford relief from the symptoms of the disease or condition being treated.

Physicians, pharmacologists, and other skilled artisans are able to determine the most therapeutically effective treatment regimen, which will vary from patient to patient. The potency of botulinum toxin and its duration of action means that doses are administered on an infrequent basis. Skilled artisans are also aware that the treatment regimen must be commensurate with questions of safety and the effects produced by the toxin.

Typically, the type E neurotoxin complex is suspended in a physiologically acceptable solution, such as normal saline, and is administered by an intramuscular injection. Prior to injection, careful consideration is given to the anatomy of the muscle group, in an attempt to inject the toxin complex into the area with the highest concentration of neuromuscular junctions. If the muscle mass is not very great, the injection can be performed with extremely fine, hollow, teflon-coated needles and guided by electromyography. The position of the needle in the muscle should be confirmed prior to injection of the toxin, and general anesthesia, local anesthesia, or other sedation may be used at the discretion of the attending physician, according to the age and particular needs of a given patient and the number of sites to be injected.

Claim 1 of 26 Claims

What is claimed is:

1. A substantially pure polypeptide complex comprising a Clostridium botulinum neurotoxin and one or more Clostridium botulinum type E neurotoxin associated polypeptides selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO:4, a polypeptide comprising the amino acid sequence of SEQ ID NO:3, a polypeptide comprising the amino acid sequence of SEQ ID NO:2, a polypeptide comprising the amino acid sequence of SEQ ID NO:1, and a polypeptide comprising the amino acid sequence of SEQ ID NO:5.




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