Title: Proteins within the type
E botulinum neurotoxin complex
United States Patent: 7,431,935
Issued: October 7, 2008
Inventors: Singh; Bal Ram
(Dartmouth, MA), Zhang; Zhong (New Bedford, MA)
Assignee: University of
Massachusetts (Boston, MA)
Appl. No.: 10/766,283
Filed: January 27, 2004
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.
Description of the
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
Clostridium 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,
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, see Original Patent).
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
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.
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 1118, 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 full-length 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 naturally-occurring
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, see Original Patent),
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
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, anti-idiotype (anti-Id) antibodies, and epitope-binding 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 Calmette-Guerin)
and Corynebacterium parvum.
In addition, antibodies according to the invention can be monoclonal
antibodies 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.
Nature 256:495, 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 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').sub.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').sub.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 anti-type 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 nerve-lumbrical
muscle, and chick ciliary ganglion-iris 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
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 optic-based
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 two-step sandwich immunoassay.
Antibody-bound 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 II, 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 fiber-bound
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 fiber-bound 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 radio-opaque
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, beta-galactosidase, 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 NaHCO.sub.3). 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 3.0.degree. C. 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 desired 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 antipsychotic 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 LD.sub.50 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 15 Claims
1. A method of treating a patient who is
suffering from undesirable muscle contraction that results from
exaggerated release of acetylcholine from presynaptic nerve terminals, the
method comprising administering to the patient a therapeutically effective
amount of a composition comprising 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, wherein
the composition is administered in an amount sufficient to reduce
acetylcholine release from presynaptic nerve terminals.
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