U.S. patent application number 11/538503 was filed with the patent office on 2009-02-12 for botulinum toxin for treating postherpetic neuralgia.
This patent application is currently assigned to ALLERGAN, INC.. Invention is credited to PATRICIA S. WALKER.
Application Number | 20090041805 11/538503 |
Document ID | / |
Family ID | 24934520 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090041805 |
Kind Code |
A1 |
WALKER; PATRICIA S. |
February 12, 2009 |
BOTULINUM TOXIN FOR TREATING POSTHERPETIC NEURALGIA
Abstract
Methods for treating conditions in an animal or human subject.
The conditions may be pain, skeletal muscle conditions, smooth
muscle conditions, glandular conditions and cosmetic conditions.
The methods comprise the step of administering a Clostridium
neurotoxin component or Clostridium neurotoxin component encoding
DNA to the subject using a needleless syringe.
Inventors: |
WALKER; PATRICIA S.;
(Irvine, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
ALLERGAN, INC.
Irvine
CA
|
Family ID: |
24934520 |
Appl. No.: |
11/538503 |
Filed: |
October 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10051952 |
Jan 17, 2002 |
7255865 |
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11538503 |
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09730237 |
Dec 5, 2000 |
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10051952 |
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Current U.S.
Class: |
424/239.1 ;
424/490 |
Current CPC
Class: |
C07K 14/33 20130101;
A61K 38/4893 20130101; A61P 1/00 20180101; A61P 21/00 20180101;
A61K 39/00 20130101; A61P 13/10 20180101; A61P 29/00 20180101; A61P
27/02 20180101; A61P 17/00 20180101 |
Class at
Publication: |
424/239.1 ;
424/490 |
International
Class: |
A61K 39/08 20060101
A61K039/08; A61K 9/16 20060101 A61K009/16 |
Claims
1-35. (canceled)
36. A method for treating pain associated with postherpetic
neuralgia in a patient in need thereof, the method comprising the
step of locally administering a therapeutically effective amount of
a botulinum toxin in powder form to a patient using a needleless
injector, wherein the pain associated with the postherpetic
neuralgia is thereby alleviated within 1 to 7 days of the local
administration of the botulinum toxin to the patient.
37. The method of claim 36, wherein the needless injector has an
elongated tubular nozzle and the needleless injector is connected
to or connectable to means for producing a gas flow.
38. The method of claim 37, wherein means for producing gas flow is
an energizing means.
39. The method of claim 37, wherein the gas flow is supersonic.
40. The method of claim 38, wherein the energizing means is a
compressed gas tank containing an inert compressed gas.
41. The method of claim 40, wherein the inert compressed gas is
helium.
42. The method of claim 36, wherein the botulinum toxin is
administered with a carrier made of a solid material, the solid
material selected from the group consisting of gold, platinum,
tungsten and an ice crystal.
43. The method of claim 36, wherein the pain associated with
postherpetic neuralgia is localized to an abdominal region of the
patient.
44. The method of claim 36, wherein the botulinum toxin is selected
from the group consisting of botulinum toxin type A, B, C.sub.1, D,
E, F and G.
45. A method for treating pain associated with postherpetic
neuralgia in a patient in need thereof, the method comprising the
step of locally administering a therapeutically effective amount of
botulinum toxin type A or B in powder form intradermally to a
patient using a needleless injector connected to or connectable to
means for producing a supersonic gas flow, wherein the pain
associated with the postherpetic neuralgia is thereby alleviated
within 1 to 7 days.
46. The method of claim 45, wherein a duration of pain alleviation
is provided that lasts from about 7 to about 27 months.
47. The method of claim 45, wherein pain associated with
postherpetic neuralgia is in the thorax of the patient.
48. The method of claim 45, wherein pain associated with
postherpetic neuralgia is localized to the abdomen of the patient
and the step of locally administering the botulinum toxin is to the
abdomen of the patient.
49. The method of claim 45, wherein the therapeutically effective
amount of the botulinum toxin type A administered to the patient is
between about 0.05 U/kg to about 2 U/kg.
50. A method for treating pain associated with postherpetic
neuralgia in a patient in need thereof, the method comprising the
step of administering locally a therapeutically effective amount of
botulinum toxin type A in powder form, using a needleless injector
connected to or connectable to energizing means for producing a
supersonic gas flow to a patient having healed lesions and pain
localized to an abdominal region, wherein administration of the
therapeutically effective amount of botulinum toxin type A in
powder form is intradermal and into the abdominal region, wherein
pain localized to the abdominal region is thereby alleviated within
1 to 7 days of the local administration of the botulinum toxin.
51. The method of claim 50, wherein the botulinum toxin is
administered with a carrier made of a solid material, wherein the
solid material is selected from the group consisting of gold,
platinum, tungsten and an ice crystal.
52. The method of claim 51, wherein the carrier has a diameter less
than about 10 millimeters.
53. The method of claim 50, wherein a duration of the pain
alleviation is provided that last from about 7 to about 27 months.
Description
RELATED APPLICATION
[0001] The present application is continuation in part of
application Ser. No. 09/730,237 filed Dec. 5, 2000, the disclosure
of which is incorporated, in its entirety, herein by reference.
BACKGROUND OF THE INVENTION
[0002] Botulinum Toxin {tc "Botulinum Toxin" \l 5}
[0003] The anaerobic, Gram positive bacterium Clostridium 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.
[0004] 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.sub.50 in mice. One unit (U) of
botulinum toxin is defined as the amount of toxin that kills 50% of
mice upon intraperitoneal injection into female Swiss webster mice
weighing 18-20 grams each. Seven immunologically distinct botulinum
neurotoxins have been characterized, these being respectively
botulinum neurotoxin serotypes, A, B, C, D, E, F and G each of
which is distinguished by neutralization with serotype-specific
antibodies. 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).
[0005] Additionally, BoNt/B has been determined to be non-toxic in
primates at a dose of 480 U/kg which is about 12 times the primate
LD.sub.50 for BoNt/A. Botulinum toxin apparently binds with high
affinity to cholinergic motor neurons, is translocated into the
neuron and blocks the release of acetylcholine.
[0006] 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.
[0007] 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.sub.1
(BoNT/C.sub.1) has been shown to cleave both syntaxin and SNAP-25.
These differences in mechanism of action may affect the relative
potency and/or duration of action of the various botulinum toxin
serotypes.
[0008] 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 pre-synaptic 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.sub.C, appears to be important for targeting of
the toxin to the cell surface.
[0009] 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.sub.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.
[0010] 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.
[0011] 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, are 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.
[0012] 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.
[0013] 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.sub.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.
[0014] It has been reported that BoNt/A has been used in clinical
settings as follows:
[0015] (1) about 75-125 units of BOTOX.RTM..sup.1 per intramuscular
injection (multiple muscles) to treat cervical dystonia;
.sup.1Available from Allergen, Inc., of Irvine, Cal. under the
tradename BOTOX.RTM..
[0016] (2) 5-10 units of BOTOX.RTM. 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);
[0017] (3) about 30-80 units of BOTOX.RTM. to treat constipation by
intrasphincter injection of the puborectalis muscle;
[0018] (4) about 1-5 units per muscle of intramuscularly injected
BOTOX.RTM. 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.
[0019] (5) to treat strabismus, extraocular muscles have been
injected intramuscularly with between about 1-5 units of
BOTOX.RTM., 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).
[0020] (6) to treat upper limb spasticity following stroke by
intramuscular injections of BOTOX.RTM. into five different upper
limb flexor muscles, as follows: [0021] (a) flexor digitorum
profundus: 7.5 U to 30 U Available from Allergan, Inc., of Irvine,
Calif. under the tradename BOTOX.RTM.. [0022] (b) flexor digitorum
sublimus: 7.5 U to 30 U [0023] (c) flexor carpi ulnaris: 10 U to 40
U [0024] (d) flexor carpi radialis: 15 U to 60 U [0025] (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 subject
receives from 90 U to 360 U of upper limb flexor muscle BOTOX.RTM.
by intramuscular injection at each treatment session.
[0026] 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.RTM. and Dysport.RTM.) 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.sub.50 values were calculated from dose
response curves. Additional mice were given intramuscular
injections to determine LD.sub.50 doses. The therapeutic index was
calculated as LD.sub.50/ED.sub.50. Separate groups of mice received
hind limb injections of BOTOX.RTM. (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.RTM.). Peak muscle weakness and duration were
dose related for all serotypes. DAS ED.sub.50 values (units/kg)
were as follows: BOTOX.RTM.: 6.7, Dysport.RTM.: 24.7, BoNt/B: 27.0
to 244.0, BoNT/F: 4.3. BOTOX.RTM. had a longer duration of action
than BoNt/B or BoNt/F. Therapeutic index values were as follows:
BOTOX.RTM.: 10.5, Dysport.RTM.: 6.3, BoNt/B: 3.2. Water consumption
was greater in mice injected with BoNt/B than with BOTOX.RTM.,
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.RTM. 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.RTM. and
Dysport.RTM.) 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.RTM., possibly because of the higher
protein load injected to achieve an effective dose of BoNt/B.
[0027] 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.
[0028] Acetylcholine
[0029] 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.
[0030] 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.
[0031] 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.
[0032] Acetylcholine is released from cholinergic neurons when
small, clear, intracellular vesicles fuse with the pre-synaptic
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.
[0033] 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.
[0034] Botulinum toxin has been shown to be effective in treating a
number of conditions. For example, botulinum toxin may alleviate
hyperhidrosis for up to 11 months. 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.
[0035] Skin gene therapy is an effective method to directly deliver
and transiently express genes in the skin. Several different
delivery methods have been successfully used in recent years. Three
of these delivery methods are needleless injection, topical gene
delivery and direct gene delivery by injection using a needle.
[0036] In needleless injection delivery methods, microprojectile
carrier particles may be coated with DNA encoding the desired gene
and then discharged into the skin from an external delivery device.
Depending on the discharge velocity and the distance from the
injection site, the drug particles penetrate through the stratum
corneum to different layers of the epidermis, dermis and underlying
muscle. As the DNA-coated microprojectiles penetrate through
epidermal and dermal cells, or are deposited in these cells, DNA is
released and the encoded genes can be expressed. The cells
potentially targeted by these drug particles in the epidermis
include, but are not limited to keratinocytes, melanocytes and
Langerhans cells. In the dermis fibroblasts, endothelial cells,
adipocytes and dermal dendritic cells may be potential targets. If
the microprojectiles penetrate through the dermis, underlying
muscle cells could be targeted. One important aspect of this
mechanism of delivery is that the DNA is directly delivered into
the cell by penetration. Therefore, the issue of skin cell's
ability to uptake DNA is not relevant. This means that all skin
cells exposed to the DNA coated microprojectiles are potential
targets.
[0037] In topical gene delivery, DNA can be applied to the skin as
either a liposomal-DNA mixture or as an uncoated DNA for
epicutaneous transfer into the epidermis. Primarily epidermal cells
would be targeted with this delivery method. However, other cells
may be targeted with needles
[0038] Gene delivery by injection with a needle is another method
of gene delivery to the skin. With this method, the DNA is
typically introduced directly into the dermis. Both epidermal and
dermal cells have access to and can express the DNA.
Electroinjection and electroporation methods of delivery are
modifications of the direct injection method where a needle is
used. These two methods can result in a higher level of gene
expression than conventional injection using a needle. After
intradermal injection of the DNA, electric pulses are applied to
the injected area by electrodes for improved cellular uptake.
[0039] All these methods of gene delivery can be used for
expression of botulinum toxin encoding DNA.
SUMMARY OF THE INVENTION
[0040] The present invention provides new and improved methods for
the injection of botulinum toxin into an animal or human subject.
The present invention also provides for methods for injecting
botulinum toxin encoding DNA into an animal or human subject.
[0041] In accordance with the present invention, there are provided
methods for treating a condition in an animal or human subject.
These conditions may comprise pain, skeletal muscle conditions,
smooth muscle conditions and glandular conditions. Botulinum toxins
are also used for cosmetic purposes. The methods may comprise a
step of administering a Clostridium neurotoxin component to the
subject using a needleless syringe.
[0042] In one embodiment, the neurotoxin component is administered
with a carrier, wherein the neurotoxin is coated on the carrier. In
another embodiment, the neurotoxin is mixed with the carrier. The
carrier may comprise a dense material, for example, gold, platinum,
tungston or ice.
[0043] Still further in accordance with the present invention, the
condition treated may be spasmodic dysphonia, laryngeal dystonia,
oromandibular dysphonia, lingual dystonia, cervical dystonia, focal
hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid
disorder, cerebral palsy, focal spasticity, spasmodic colitis,
neurogenic bladder, anismus, limb spasticity, tics, tremors,
bruxism, anal fissure, achalasia, fibromyalgia, dysphagia,
lacrimation, hyperhydrosis, excessive salivation, excessive
gastrointestinal secretions, excessive mucous secretion, pain from
muscle spasms, headache pain, brow furrows and skin wrinkles.
[0044] Still further in accordance with the present invention, the
neurotoxin component may be administered to the skin. The skin may
comprise an epidermis layer, a dermis layer and a hypodermis
layer.
[0045] In one embodiment, the neurotoxin component is administered
to one or more layers of a skin where a nerve is located.
[0046] In another embodiment, the neurotoxin component is
administered to a skin and substantially to a muscle tissue.
[0047] In still another embodiment, the neurotoxin component is
administered to a muscle tissue.
[0048] Still further in accordance with the present invention, the
neurotoxin component may be a difficile toxin, a butyricum toxin a
tetani toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, or G
or a variant thereof.
[0049] Still further in accordance with the present invention, the
neurotoxin component may comprise a targeting component, a
therapeutic component and a translocation component.
[0050] In one embodiment, the targeting component binds to a cell,
for example, a nerve cell. In one embodiment the targeting
component binds to a pre-synaptic nerve terminal. The pre-synaptic
nerve terminal may belong to a cholinergic neuron.
[0051] The targeting component may comprise, for example, a
carboxyl end segment of a heavy chain of a butyricum toxin, a
tetani toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, G or
a variant thereof.
[0052] In one embodiment, the therapeutic component substantially
interferes with exocytosis from a cell, for example, interfering
with the release of neurotransmitters from a neuron or its
terminals.
[0053] The therapeutic component may comprise, for example, a light
chain of a butyricum toxin, a tetani toxin or a botulinum toxin
type A, B, C.sub.1, D, E, F, G or a variant thereof.
[0054] In one embodiment, the translocation component facilitates
transfer of at least a part of the neurotoxin component into the
cytoplasm of a target cell.
[0055] The translocation component may comprise, for example, an
amino end fragment of a heavy chain of a butyricum toxin, a tetanti
toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, G or a
variant thereof.
[0056] The targeting component may comprise, for example, a
carboxyl end fragment of a heavy chain of a botulinum toxin type A,
the therapeutic component may comprise a light chain of a botulinum
toxin type A and the translocation component may comprise an amine
end fragment of a heavy chain of botulinum toxin type A.
[0057] Still further in accordance with the present invention, the
neurotoxin component may be recombinantly produced.
[0058] Still further in accordance with the present invention, are
methods for expressing a recombinant DNA sequence encoding a
Clostridium neurotoxin component in a cell of an animal in situ.
The cell may be, for example, a skin cell, a muscle cell or a nerve
cell.
[0059] In one embodiment, the DNA is administered to the animal by
injection. For example, the injection may be by needleless
injection.
[0060] Still further in accordance with the present invention, the
DNA encoding neurotoxin component may be a difficile toxin, a
butyricum toxin, a tetani toxin or a botulinum toxin type A, B,
C.sub.1, D, E, F, or G or a variant thereof.
[0061] In one embodiment, the DNA encoding neurotoxin component
comprises a targeting component, a therapeutic component and a
translocation component.
[0062] Still further in accordance with the present invention, the
targeting component may bind to a cell, for example, a nerve cell.
In one embodiment, the targeting component binds to a pre-synaptic
nerve terminal. The pre-synaptic nerve terminal may belong to a
cholinergic neuron.
[0063] Still further in accordance with the present invention, the
targeting component may comprise a carboxyl end segment of a heavy
chain of a butyricum toxin, a tetani toxin or a botulinum toxin
type A, B, C.sub.1, D, E, F, G or a variant thereof.
[0064] Still further in accordance with the present invention, the
therapeutic component may substantially interfere with exocytosis
from a cell, for example, interfering with the release of
neurotransmitters from a neuron or its terminals.
[0065] The therapeutic component may comprise, for example, a light
chain of a butyricum toxin, a tetani toxin or a botulinum toxin
type A, B, C.sub.1, D, E, F, G or a variant thereof.
[0066] Still further in accordance with the present invention, the
translocation component may facilitate transfer of at least a part
of the neurotoxin component into the cytoplasm of a target
cell.
[0067] The translocation component may comprise, for example, an
amino end fragment of a heavy chain of a butyricum toxin, a tetnus
toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, G or a
variant thereof.
[0068] 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 botulinum toxin type A.
[0069] Still further in accordance with the present invention, the
neurotoxin component may be recombinantly produced.
[0070] Still further in accordance with the present invention, are
compositions that may comprise a carrier and a Clostridial
neurotoxin component, the composition may be useful for delivery of
said neurotoxin component to a cell of an animal in situ.
[0071] The carrier may be a dense material for example gold,
tungsten, platinum or ice crystal.
[0072] Still further in accordance with the present invention, the
neurotoxin component may be a difficile toxin, a butyricum toxin a
tetani toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, or G
or a variant thereof.
[0073] Still further in accordance with the present invention, the
neurotoxin component comprises a targeting component, a therapeutic
component and a translocation component.
[0074] Still further in accordance with the present invention, the
targeting component may bind to a cell, for example, a nerve cell.
In one embodiment, the targeting component binds to pre-synaptic
nerve terminal. The pre-synaptic nerve terminal may belong to a
cholinergic neuron.
[0075] Still further in accordance with the present invention, the
targeting component may comprise a carboxyl end segment of a heavy
chain of a butyricum toxin, a tetani toxin or a botulinum toxin
type A, B, C.sub.1, D, E, F, G or a variant thereof.
[0076] In one embodiment, the therapeutic component substantially
interferes with exocytosis from a cell, for example, interfering
with the release of neurotransmitters from a neuron or its
terminals.
[0077] The therapeutic component may comprise, for example, a light
chain of a butyricum toxin, a tetani toxin or a botulinum toxin
type A, B, C.sub.1, D, E, F, G or a variant thereof.
[0078] Still further in accordance with the present invention, the
translocation component may facilitate transfer of at least a part
of the neurotoxin component into the cytoplasm of a target
cell.
[0079] The translocation component may comprise, for example, an
amino end fragment of a heavy chain of a butyricum toxin, a tetanti
toxin or a botulinum toxin type A, B, C.sub.1, D, E, F, G or a
variant thereof.
[0080] 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 botulinum toxin type A.
[0081] Still further in accordance with the present invention, is a
method to treat a condition in a subject comprising administering a
therapeutically effective amount of DNA encoding a Clostridial
neurotoxin component to a cell of an animal, for example, a human
subject in situ. The cell may be, for example, a skin cell, a
muscle cell or a nerve cell.
[0082] In one embodiment, the DNA is administered to the subject by
injection. For example, the injection may be by needleless
injection.
[0083] Still further in accordance with the present invention, the
condition may comprise pain, skeletal muscle conditions, smooth
muscle conditions and/or glandular conditions. In addition, DNA
encoding a Clostridial neurotoxin may be administered to a subject
for cosmetic purposes. For example, the condition may be spasmodic
dysphonia, laryngeal dystonia, oromandibular dysphonia, lingual
dystonia, cervical dystonia, focal hand dystonia, blepharospasm,
strabismus, hemifacial spasm, eyelid disorder, cerebral palsy,
focal spasticity, spasmodic colitis, neurogenic bladder, anismus,
limb spasticity, tics, tremors, bruxism, anal fissure, achalasia,
fibromyalgia, dysphagia, lacrimation, hyperhydrosis, excessive
salivation, excessive gastrointestinal secretions, excessive mucous
secretion, pain from muscle spasms, headache pain, brow furrows and
skin wrinkles.
[0084] Still further in accordance with the present invention, are
methods for immunization which include administering an effective
amount of DNA encoding a Clostridial neurotoxin component to a
tissue, for example, the skin, of a subject. The administering may
be by injection, for example, by needleless injection.
[0085] Still further in accordance with the present invention, are
compositions comprising a carrier and a DNA sequence encoding a
Clostridial neurotoxin which may be useful for delivery of the DNA
to a cell of a an animal or human subject in situ.
[0086] Still further in accordance with the present invention, the
DNA encoding a neurotoxin may encode, for example, botulinum type
A, B, C.sub.1, D, E, F, G or mixtures thereof or combinations
thereof.
[0087] Any and all features described herein and combinations of
such features are included within the scope of the invention
provided that such features of any such combination are not
mutually exclusive.
[0088] These and other aspects and advantages of the present
invention are apparent in the following detailed description and
claims.
DEFINITIONS
[0089] Before proceeding to describe the present invention, the
following definitions are provided and apply herein.
[0090] "Affected skin area" means an area which may be in the
vicinity of the area to be treated by needleless injection, for
example, an area of skin at or near an area of skin with excessive
sweating.
[0091] "Drug particle" means a drug, for example, a neurotoxin or,
for example, a DNA sequence encoding a neurotoxin, alone, or in
combination with one or more other substances, for example,
gold.
[0092] "Without using a needle" or "needleless injection" means
injecting a measurable amount of substance, for example, a carrier
coated with a botulinum toxin without the use of a standard
needle.
[0093] "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.
[0094] "H.sub.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.
[0095] "H.sub.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, pre-synaptic binding to motor neurons.
[0096] "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.
[0097] "Neurotoxin" means a chemical entity that is capable of
interfering with the functions of a neuron. For example, a
neurotoxin may interfere with the transmission of an electrical
signal from a nerve cell to its target.
[0098] The target may be, for example, another nerve cell, a tissue
or an organ. The "neurotoxin" may be naturally occurring or
other.
[0099] "Variant" means a chemical entity which is slightly
different from a parent chemical entity but which still has a
biological effect similar, or substantially similar to the
biological effect of the chemical entity. The biological effect of
the variant may be substantially the same or better than that of
the parent. For example, a variant neurotoxin component may have
one or more amino acid substitutions, amino acid modifications,
amino acid deletions and/or amino acid additions. An amino acid
substitution may be conservative or non-conservative, as is well
understood in the art. In addition, variants of neurotoxin
components may include neurotoxin components that have modified
amino acid side chains, as is well known in the art. Variants may
also include fragments.
[0100] An example of a variant neurotoxin component may comprise a
variant light chain of a botulinum toxin having one or more amino
acids substituted, modified, deleted and/or added. This variant
light chain may have the same or better ability to prevent
exocytosis, for example, the release of neurotransmitter vesicles.
Additionally, the biological effect of a variant may be decreased
compared to the parent chemical entity. For example, a variant
light chain of a botulinum toxin type A having an amino acid
sequence removed may have a shorter biological persistence than
that of the parent (or native) botulinum toxin type A light
chain.
[0101] "Fragment" means an amino acid or nucleotide sequence that
comprises 1% or more of the parent amino acid or nucleotide
sequence. For example, a fragment of botulinum toxin type A
comprises 1% or more of the amino acid sequence of botulinum type
A.
DETAILED DESCRIPTION OF THE INVENTION
[0102] Methods for administering neurotoxins, and DNA encoding
neurotoxins, to animals, for example, humans are disclosed herein.
In one broad embodiment, methods for administering neurotoxins
include a step of administering a neurotoxin without using a
needle. In another broad embodiment, there are provided methods of
administering a DNA nucleotide sequence which encodes a neurotoxin
to an animal or human subject.
[0103] Using these methods of administration, botulinum toxin can
be used to treat a variety of conditions that are benefited by
botulinum toxin treatment. For example, spasmodic dysphonia,
laryngeal dystonia, oromandibular dysphonia, lingual dystonia,
cervical dystonia, focal hand dystonia, blepharospasm, strabismus,
hemifacial spasm, eyelid disorder, cerebral palsy, focal
spasticity, spasmodic colitis, neurogenic bladder, anismus, limb
spasticity, tics, tremors, bruxism, anal fissure, achalasia,
fibromyalgia, dysphagia, lacrimation, hyperhydrosis, excessive
salivation, excessive gastrointestinal secretions, as well as other
secretory disorders, pain from muscle spasms, headache pain, brow
furrows and skin wrinkles and other muscle tone disorders, and
other disorders, characterized by involuntary movements of muscle
groups may be treated using the present methods of administration.
Further, the methods of administration of the present invention are
useful for immunization against a neurotoxin.
[0104] 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.
[0105] Just below 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.
[0106] Accordingly, the method of this invention delivers a
neurotoxin, or DNA encoding a neurotoxin, to a tissue of an animal
or a human subject. In one embodiment, the drug is delivered to the
layer of the skin in which nerve terminals are found. For example,
delivery is to the dermis layer. In another embodiment, delivery is
to at least one layer of the skin and substantially to tissues
beneath. For example, the administration to the dermis layer of the
skin and to the subcutaneous layer. In another embodiment, delivery
is to the skin and to muscle tissues beneath. In still another
embodiment, delivery is substantially to the muscle tissue.
[0107] The administration of a composition comprising a carrier and
a neurotoxin component and/or DNA encoding a neurotoxin component
according to the invention may be accomplished through the use of a
needleless injector.
[0108] Needleless injectors and their use are well 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 comprises 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 mediums to high velocity toward a skin surface and into
the skin surface. Such a device may be purchased from PowderJect
Pharmaceuticals, Oxford, UK. In one embodiment, the gas pressure
provided must be sufficient to discharge the compositions 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 compositions to a target site, for
example the dermis, but not so great as to damage the skin surface,
for example the epithelium. In another embodiment, the gas pressure
is sufficient to deliver the compositions to the dermis layer, but
not to the layers below, for example the subcutaneous layer and/or
the muscle tissues. In another embodiment, the gas pressure
provided must be sufficient to discharge the drug particles into a
targeted site, for example the dermis and/or substantially to the
muscle tissue below, but not so great as to damage the skin
surface.
[0109] Advantages for using a needleless injector according to the
present invention include, for example, an optimal delivery to a
specific tissue layer, for example the dermis layer. Furthermore,
in the case where the delivery is to the dermis and not the muscle
tissues, the treatment may 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 conditions with little pain or skin damage such
as bruising or bleeding.
[0110] A drug particle may comprise a neurotoxin component and a
carrier component. The neurotoxin may include a targeting
component, a therapeutic component and a translocation component.
The targeting component may bind to a pre-synaptic nerve terminal,
for example a pre-synaptic 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
botulinum toxin type A, B, C.sub.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.
[0111] The therapeutic component may substantially interfere with
exocytosis from a cell, for example, interfering 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 botulinum toxin type A, B, C.sub.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.
[0112] 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 botulinum toxin type A, B, C.sub.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.
[0113] 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.RTM..
[0114] 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 DNA
sequences which encode at least one of the neurotoxin components,
for example the therapeutic component, translocation component
and/or targeting component. The DNA encoding the neurotoxin is
inserted into an expression vector with compatible cohesive end
terminals that will allow for the annealing and subsequent ligation
of the neurotoxin encoding DNA insert. After ligation the
recombinant DNA molecules are transformed into a host cell such as
E. coli. Transformants are screened for by, for example, blue-white
screening, as is known in the art. After identification of a
recombinant vector containing the appropriate insert by for
example, restriction digest analysis and/or nucleotide sequence
analysis, the recombinant neurotoxin is expressed using either a
constituitive or inducible promoter depending on the type of
expression vector used. The recombinant protein produced by the
expression system can be isolated using conventional techniques.
For example, if an expression vector which produces a
polyhis-factor Xa fusion protein is used, the protein can be first
isolated on a metal containing column, such as a nickel, and then
cleaved with factor Xa to release the neurotoxin molecule. Many
variations for producing neurotoxins by recombinant methodologies
exist and are familiar to those skilled in the art. For example,
yeast, mammalian or insect cell systems may be used to produce
recombinant neurotoxin proteins.
[0115] The recombinant protein 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.sub.N, of a botulinum toxin type B
(the translocation component), and an H.sub.C of 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.
[0116] 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 di-chain form in which the two chains
remain covalently linked through the intrachain disulfide bond.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] In another embodiment, a DNA nucleotide sequence encoding a
neurotoxin is injected into an animal or human subject. For
example, the DNA nucleotide sequence may be that of botulinum toxin
type A (SEQ. ID. #1), type B (SEQ. ID. #2 and #3), type C.sub.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. In one embodiment, the injected DNA nucleotide
sequence encodes a Clostridial toxin or a variant of a Clostridial
toxin. In one embodiment the nucleotide sequence encodes a
botulinum toxin type A. In another embodiment the DNA nucleotide
sequence encodes a fragment of a neurotoxin. For example, the DNA
sequence may encode a therapeutic component, for example, a light
chain of a botulinum toxin.
[0121] Injection of the DNA nucleotide sequence may be, for
example, to treat a condition in an animal, for example a human.
Injection of a DNA nucleotide sequence encoding a Clostridial toxin
may also be used to immunize an animal or human subject.
[0122] Injection of the DNA nucleotide sequence may also be used
for research purposes. For example, these methods may be used to
examine the expression of Clostridial genes inside an animal cell
in situ. Also, for example, activity of a neurotoxin inside of an
animal cell in situ may be studied using these methods.
[0123] A neurotoxin or DNA sequence encoding a neurotoxin may be
injected alone, or in combination with other drugs and/or agents.
In either case, the neurotoxin or DNA sequence encoding a
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 DNA
nucleotide sequence may be injected in accordance with this
invention, it is preferable that the injected DNA 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.
[0124] 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 one
embodiment, the carrier is less than about 10 mm, more preferably
less than about 5 mm, even more preferably less than about 3 mm.
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 mm, wherein the neurotoxin protein 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 mm 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 mm. In one
embodiment, neurotoxin is coated onto ice crystal and/or gold
carriers to form drug particles. In a another embodiment, botulinum
toxin type A is coated onto ice crystals and/or gold carriers to
form drug particles to be used in accordance with this
invention.
[0125] 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.RTM. and Triton.RTM. surfactants (Union Carbide Chemicals
and Plastics, Danbury, Conn.), polyoxyethylenesorbitans, for
example, TWEEN.RTM. surfactants (Atlas Chemical Industries,
Wilmington, Del.), polyoxyethylene ethers, for example, Brij.RTM.,
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.
[0126] In one broad embodiment, the step of administering a
neurotoxin or DNA sequence encoding a neurotoxin 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; and/or administering anticholinergics
orally or systemically.
[0127] The following examples demonstrate how various conditions
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
subject variables including size, weight, age, and responsiveness
to therapy.
[0128] 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 Post Surgical Myofacial Pain Syndrome
[0129] An unfortunate 36 year old woman has a 15 year history of
temporomandibular joint disease and chronic pain along the masseter
and temporalis muscles. Fifteen years prior to evaluation she noted
increased immobility of the jaw associated with pain and jaw
opening and closing and tenderness along each side of her face. The
left side is originally thought to be worse than the right. She is
diagnosed as having temporomandibular joint (TMJ) dysfunction with
subluxation of the joint and is treated with surgical orthoplasty
meniscusectomy and condyle resection.
[0130] She continues to have difficulty with opening and closing
her jaw after the surgical procedures and for this reason, several
years later, a surgical procedure to replace prosthetic joints on
both sides is performed. After the surgical procedure, progressive
spasms and deviation of the jaw ensues. Further surgical revision
is performed subsequent to the original operation to correct
prosthetic joint loosening. The jaw continues to exhibit
considerable pain and immobility after these surgical procedures.
The TMJ remained tender as well as the muscle itself. There are
tender points over the temporomandibular joint as well as increased
tone in the entire muscle. She is diagnosed as having post-surgical
myofascial pain syndrome and is treated with a needleless injection
of 20 U of botulinum type A neurotoxin into the skin covering the
masseter and temporalis muscles.
[0131] Several days after the injections she noted substantial
improvement in her pain and reports that her jaw feels looser. This
gradually improves over a 2 to 3 week period in which she notes
increased ability to open the jaw and diminishing pain. The patient
states that the pain is better than at any time in the last 4
years. The improved condition persists for up to 27 months after
the original injection of the modified neurotoxin.
EXAMPLE 2
Peripheral Administration of a Modified Neurotoxin to Treat
"Shoulder-Hand Syndrome"
[0132] Pain in the shoulder, arm, and hand can develop, with
muscular dystrophy, osteoporosis, and fixation of joints. While
most common after coronary insufficiency, this syndrome may occur
with cervical osteoarthritis or localized shoulder disease, or
after any prolonged illness that requires the patient to remain in
bed.
[0133] A 46 year old woman presents a shoulder-hand syndrome type
pain. The pain is particularly localized at the deltoid region. The
patient is treated by a needleless injection of between about 0.05
U/kg to about 2 U/kg of a modified neurotoxin cutaneously to the
shoulder, preferably the neurotoxin is botulinum type A. The
particular dose as well as the frequency of administrations depends
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within 1-7 days after modified
neurotoxin administration the patient's pain is substantially
alleviated. The duration of the pain alleviation is from about 7 to
about 27 months.
EXAMPLE 3
Peripheral Administration of a Modified Neurotoxin to Treat
Postherpetic Neuralgia
[0134] Postherpetic neuralgia is one of the most intractable of
chronic pain problems. Patients suffering this excruciatingly
painful process often are elderly, have debilitating disease, and
are not suitable for major interventional procedures. The diagnosis
is readily made by the appearance of the healed lesions of herpes
and by the patient's history. The pain is intense and emotionally
distressing. Postherpetic neuralgia may occur any where, but is
most often in the thorax.
[0135] A 76 year old man presents a postherpetic type pain.
[0136] The pain is localized to the abdomen region. The patient is
treated by a needleless injection of between about 0.05 U/kg to
about 2 U/kg of a modified neurotoxin intradermally to the abdomen,
preferably the modified neurotoxin is BoNT/E fused with a
leucine-based motif. The particular dose as well as the frequency
of administrations depends upon a variety of factors within the
skill of the treating physician, as previously set forth. Within
1-7 days after modified neurotoxin administration the patient's
pain is substantially alleviated. The duration of the pain
alleviation is from about 7 to about 27 months.
EXAMPLE 4
Peripheral Administration of a Modified Neurotoxin to Treat
Inflammatory Pain
[0137] A patient, age 45, presents an inflammatory pain in the
chest region. The patient is treated by a needleless injection of
between about 0.05 U/kg to about 2 U/kg of a botulinum neurotoxin
type A intramuscularly to the chest. The particular dose as well as
the frequency of administrations depends upon a variety of factors
within the skill of the treating physician, as previously set
forth. Within 1-7 days after modified neurotoxin administration the
patient's pain is substantially alleviated. The duration of the
pain alleviation is from about 7 to about 27 months.
EXAMPLE 5
{tc "Example 5"\l 4} Local Administration of a Neurotoxin to Treat
Pain Caused by Bone Fractures {tc "Local Administration of a
Neurotoxin to Treat Pain Caused by Bone Fractures" \l 3}
[0138] A patient, age 40, suffering from cervical dystonia is
treated by an needleless injection of a neurotoxin, preferably
botulinum toxin type A, at the effected area of the spine. The
amount of neurotoxin injected is between about 20 U to about 500 U.
The particular dose as well as the frequency of administrations
depends upon a variety of factors within the skill of the treating
physician, as previously set forth. Within 1-7 days after
administration the patient's pain is substantially alleviated. The
duration of pain reduction is from about 2 to about 7 months.
EXAMPLE 6
Treatment of Pain Associated with Muscle Disorder
[0139] An unfortunate 36 year old woman has a 15 year history of
temporomandibular joint disease and chronic pain along the masseter
and temporalis muscles. Fifteen years prior to evaluation she noted
increased immobility of the jaw associated with pain and jaw
opening and closing and tenderness along each side of her face. The
left side is originally thought to be worse than the right. She is
diagnosed as having temporomandibular joint (TMJ) dysfunction with
subluxation of the joint and is treated with surgical orthoplasty
meniscusectomy and condyle resection.
[0140] She continues to have difficulty with opening and closing
her jaw after the surgical procedures and for this reason, several
years later, a surgical procedure to replace prosthetic joints on
both sides is performed. After the surgical procedure progressive
spasms and deviation of the jaw ensues. Further surgical revision
is performed subsequent to the original operation to correct
prosthetic joint loosening. The jaw continues to exhibit
considerable pain and immobility after these surgical procedures.
The TMJ remained tender as well as the muscle itself. There are
tender points over the temporomandibular joint as well as increased
tone in the entire muscle. She is diagnosed as having post-surgical
myofascial pain syndrome and is treated with a needleless injection
of 15 U of botulinum toxin type A into the masseter and temporalis
muscles.
[0141] Several days after the injections she noted substantial
improvement in her pain and reports that her jaw feels looser. This
gradually improves over a 2 to 3 week period in which she notes
increased ability to open the jaw and diminishing pain. The patient
states that the pain is better than at any time in the last 4
years. The improved condition persists for up to 27 months after
the original injection of the modified neurotoxin.
EXAMPLE 7
Treatment of Pain Subsequent to Spinal Cord Injury
[0142] A patient, age 39, experiencing pain spasticity of the right
side bicep muscle is treated by needleless injection with about 1.0
U/kg of the modified neurotoxin, preferably the modified neurotoxin
is botulinum toxin type A. The particular toxin dose and site of
injection, as well as the frequency of toxin administrations depend
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within about 1 to about 7 days
after the modified neurotoxin administration, the patient's muscle
spasms are substantially reduced. The spasm alleviation persists
for up to 27 months.
EXAMPLE 8
Peripheral Administration of a Modified Neurotoxin to Treat
"Shoulder-Hand Syndrome"
[0143] A 46 year old woman presents a shoulder-hand syndrome type
pain. The pain is particularly localized at the deltoid region. The
patient is treated by a needleless injection of between about 0.05
U/kg to about 2 U/kg of botulinum type A neurotoxin subcutaneously
to the shoulder. The particular dose as well as the frequency of
administrations depends upon a variety of factors within the skill
of the treating physician, as previously set forth. Within 1-7 days
after modified neurotoxin administration the patient's pain is
substantially alleviated. The duration of the pain alleviation is
from about 7 to about 27 months.
EXAMPLE 9
Treatment of Axillary Hyperhidrosis
[0144] 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.
Iontophoresis using special axillary electrodes are also employed
in the treatment of axillary hyperhidrosis. Oral sedatives,
tranquilizers or anticholinergic drugs are sometimes used as an
adjunct.
[0145] 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.
[0146] 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.
[0147] Botulinum toxin type A coated on crystal ice particle
carrier is loaded into a needleless injector.
[0148] 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.times.15 cm.sup.2 of 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.
[0149] 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.
EXAMPLE 10
Treatment of Palmar Hyperhidrosis
[0150] 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 papillarv 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.
[0151] 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.
[0152] Botulinum toxin type A coated on crystal ice particle
carrier is loaded into a needleless injector.
[0153] 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.times.15 cm.sup.2 of 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.
[0154] 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.
EXAMPLE 11
Subcloning the BoNT/A-L Chain Gene
[0155] 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'-AAAGGCCTTTTGTTAATAAACAA-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.
[0156] Additionally, these primers introduce a stop codon at the
C-terminus of the L chain coding sequence. Chromosomal DNA from C.
botulinum (strain 63 A) serves as a template in the amplification
reaction.
[0157] The PCR amplification is performed in a 100 ml volume
containing 10 mM Tris-HCI (pH 8.3), 50 mM KCl, 1.5 mM MgCI.sub.2,
0.2 mM of each deoxynucleotide triphosphate (dNTP), 50 .mu.mol of
each primer, 200 ng of genomic DNA and 2.5 units of Taq-polymerase
(Promega). The reaction mixture is patiented to 35 cycles of
denaturation (1 minute at 94.degree. C.), annealing (2 minutes at
37.degree. C.) and polymerization (2 minutes at 72.degree. C.).
Finally, the reaction is extended for an additional 5 minutes at
72.degree. C.
[0158] The PCR amplification product is digested with Stu I and
EcoR I, purified by agarose gel electrophoresis, and ligated into
Sma I and EcoR I digested pBluescript II SK* to yield the plasmid,
pSAL. Bacterial transformants harboring this plasmid are isolated
by standard procedures. The identity of the cloned L chain
polynucleotide is confirmed by double stranded plasmid sequencing
using SEQUENASE (United States Biochemicals) according to the
manufacturer's instructions. Synthetic oligonucleotide sequencing
primers are prepared as necessary to achieve overlapping sequencing
runs. The cloned sequence is found to be identical to the sequence
disclosed by Binz, et al., in J. Biol. Chem. 265:9153 (1990), and
Thompson et al., in Eur. J. Biochem. 189:73 (1990).
[0159] Site-directed mutants designed to compromise the enzymatic
activity of the BoNT/A-L chain can also be created.
EXAMPLE 12
Expression of the Botulinum Toxin Type A-L (BoNt/A-L) Chain Fusion
Proteins
[0160] 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 mg/ml ampicillin and 2% (w/v) glucose, and grown
overnight with shaking at 30.degree. C. The overnight cultures are
diluted 1:10 into fresh L-broth containing 100 mg/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.degree. C., bacteria are
collected by centrifugation at 6,000.times.g for 10 minutes.
[0161] A small-scale SDS-PAGE analysis confirmed the presence of a
90 kDa protein band in samples derived from IPTG-induced bacteria.
This M.sub.r is consistent with the predicted size of a fusion
protein having MBP (.about.40 kDa) and BoNT/A-L chain (.about.50
kDa) components. Furthermore, when compared with samples isolated
from control cultures, the IPTG-induced clones contained
substantially larger amounts of the fusion protein.
[0162] 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 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.sub.r than 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.
[0163] The yield of intact fusion protein isolated by the above
procedure remained fully adequate for all 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.
[0164] 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 Xa. 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 13
Purification of Fusion Proteins and Isolation of Recombinant
BoNT/A-L Chains
[0165] This Example describes a method to produce and purify
wild-type recombinant BoNT/A light chains from bacterial clones.
Pellets from 1 liter cultures of bacteria expressing the wild-type
BoNT/A-L chain proteins are resuspended in column buffer [10 mM
Tris-HCI (pH 8.0), 200 mM NaCl, 1 mM EGTA and I mM DTT] containing
1 mM phenyl-methanesulfonyl fluoride (PMSF) and 10 mM benzamidine,
and lysed by sonication. The lysates are cleared by centrifugation
at 15,000.times.g for 15 minutes at 4.degree. C. Supernatants are
applied to an amylose affinity column [2.times.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-HCI (pH 8.0)
supplemented with 150 mM NaCl, 2 mM, CaCl.sub.2 and 1 mM DTT for 72
hours at 4.degree. C.
[0166] Fusion proteins are cleaved with Factor X.sub.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, CaCl.sub.2 and 1 mM DTT. Dialysis is
carried out for 24 hours at 4.degree. 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.degree. C. Total E. coli extract or the purified proteins are
solubilized in SDS sample buffer and patiented 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.
[0167] 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.
[0168] 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.
EXAMPLE 14
Evaluation of the Proteolytic Activities of Recombinant L Chains
Against a SNAP-25 Substrate
[0169] 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
NaCI and 2.5 mM CaCI.sub.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.degree. C.
The total protein concentration is determined by routine
methods.
[0170] 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., 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-dimethylpropyl) 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.
[0171] All recombinant preparations containing wild-type L chain
are dialyzed overnight at 4.degree. C. into 100 mM HEPES (pH 7.5)
containing 0.02% Lubrol and 10 mM zinc acetate before assessing
their enzymatic activities. BoNT/A, previously reduced with 20 mM
DTT for 30 minutes at 37.degree. C., as well as these dialyzed
samples, are then diluted to different concentrations in the latter
HEPES buffer supplemented with 1 mM DTT.
[0172] Reaction mixtures include 5 ml recombinant SNAP-25 substrate
(8.5 mM final concentration) and either 20 ml reduced BoNT/A or
recombinant wild-type L chain. All samples are incubated at
37.degree. C. for 1 hour before quenching the reactions with 25 ml
aqueous 2% trifluoroacetic acid (TFA) and 5 mM EDTA (Foran et al.,
Biochemistry 33:15365 (1994)). Aliquots of each sample are prepared
for SDS-PAGE and Western blotting with the polyclonal SNAP-25
antibody by adding SDS-PAGE sample buffer and boiling. Anti-SNAP-25
antibody reactivity is monitored using an ECL detection system and
quantified by densitometric scanning.
[0173] 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.
[0174] 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.sup.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 X.sub.2 to produce a
mixture containing free MBP and recombinant L chain, induced a
dose-dependent inhibition of Ca.sup.2+-stimulated release
equivalent to the inhibition caused by native BoNT/A.
EXAMPLE 15
Reconstitution of Native L Chain, Recombinant Wild-Type L Chain
with Purified H Chain
[0175] Native H and L chains are dissociated from BoNT/A (List
Biologicals Inc.; Campbell, USA) with 2 M urea, reduced with 100 mM
DTT and then purified according to established chromatographic
procedures (Kozaki et al., Japan J. Med. Sci. Biol. 34:61 (1981);
Maisey et al., Eur. J. Biochem. 177:683 (1988)). Purified H chain
is combined with an equimolar amount of either native L chain or
recombinant wild-type L chain. Reconstitution is carried out by
dialyzing the samples against a buffer consisting of 25 mM Tris (pH
8.0), 50 mM zinc acetate and 150 mM NaCl over 4 days at 4.degree.
C. Following dialysis, the association of the recombinant L chain
and native H chain to form disulfide-linked 150 kDa di-chains is
monitored by SDS-PAGE and quantified by densitometric scanning. The
proportion of di-chain 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.
EXAMPLE 16
A Study of Botulinum Neurotoxin Type A Activity When the Toxin is
Produced in Skin and Muscle cells of an Animal
[0176] Genes encoding botulinum toxin type A are expressed in the
skin and muscle cells of live mice in situ by needleless injection
of DNA coated microprojectiles into the tissues.
[0177] The DNA encoding recombinant botulinum toxin comprises two
nucleotide sequences. One sequence contains a human beta-actin
promoter fused to a DNA sequence encoding the heavy chain of
botulinum type A. The second sequence contains a human beta-actin
promoter fused to a DNA sequence encoding a light chain of
botulinum toxin. Polyadenylation signal sequences are added 3' to
the Clostridium genes. Stop codons are placed at the 5' end of each
gene to allow translation of the complete heavy chain and light
chains.
[0178] Substance particles, for example, gold particles or tungsten
particles, having a range of diameter from 1 to 3 micrometers or 2
to 5 micrometers are coated with DNA by mixing sequentially 25
microliters of gold or tungsten microprojectiles in an aqueous
slurry, 2.5 microliters of DNA (1 mg/ml), 25 microliters of
CaCl.sub.2 and 5 microliters of free base spermidine (1M). After 10
min of incubation, the microprojectiles are collected by
centrifugation and the supernatant removed. The pellet is washed
once in 70% ethanol, centrifuged and resuspended in 25 microliters
of 100% ethanol. The ethanol is allowed to evaporate from the DNA
coated microprojectiles before injection.
[0179] The DNA coated microprojectiles are administered into the
skin, or into the skin and underlying muscle tissue of the mice by
needleless injection.
[0180] Botulinum toxin gene expression is assessed by, for example,
in situ hybridization. In situ hybridization is performed 24 hours
after the injection. The muscle and skin tissues are frozen and
cryosectioned at 10-micrometer thickness. The sections are dried
onto gelatin/chrom alum-coated slides, fixed with 4%
paraformaldehyde and hybridized with .sup.35S-labeled synthetic
oligonucleotide probes complementary to the botulinum toxin mRNAs.
FITC labeled antibodies to the heavy and light chains were also
used as probes. Results showed 10 to 20% of the skin cells in the
area of injection expressed the botulinum toxin genes. While 5 to
10% of the muscle cells in the injection area expressed the
genes.
[0181] In mice where the DNA coated particles were administered to
the skin and substantially to underlying muscle tissue, a partial
paralysis of the effected muscle was noted.
EXAMPLE 17
Peripheral Administration of a Modified Neurotoxin DNA Encoding
Sequence to Treat Inflammatory Pain
[0182] A patient, age 45, presents a case of blepharospasm. The
patient is treated by a needleless injection to the skin near the
eye of between about 10 nanograms to about 5 micrograms of DNA
encoding botulinum neurotoxin plus appropriate flanking sequences,
preferably the neurotoxin is type A. Preferably, gold or tungsten
microprojectiles are coated with the DNA. The particular dose as
well as the frequency of administration depends upon a variety of
factors within the skill of the treating physician. Within 1-7 days
after modified neurotoxin administration the patient's pain is
substantially alleviated. The duration of the alleviation of
spasmotic winking is from about 7 to about 27 months.
EXAMPLE 18
Treatment of Gustatory Sweating
[0183] 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.
[0184] A 40 year old man presents a classic case of Frey's
syndrome. The area of hyperhydrosis 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.
[0185] DNA encoding botulinum toxin type A and appropriate flanking
sequences, i.e. transcription initiation and termination sequences,
is coated on a gold particle carrier. The coated carrier is loaded
into a needleless injector. The projection pressure is set so that
the drug particles may be delivered to the dermis layer of the
skin. The particular dose of the neurotoxin DNA and area of
injection, as well as the frequency of toxin administration depends
upon a variety of factors to be determined by the treating
physician, as previously set forth.
[0186] Seven days after treatment, the gustatory sweating is
measured using the Minor's iodine test. The hyperhidrotic area
shows about 93% reduction. The reduction in gustatory sweating
starts after about 72 hours and persists up to about 12 months.
[0187] 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.
Sequence CWU 1
1
1213891DNAbotulinum toxin 1atgcaatttg ttaataaaca atttaattat
aaagatcctg taaatggtgt tgatattgct 60tatataaaaa ttccaaatgt aggacaaatg
caaccagtaa aagcttttaa aattcataat 120aaaatatggg ttattccaga
aagagataca tttacaaatc ctgaagaagg agatttaaat 180ccaccaccag
aagcaaaaca agttccagtt tcatattatg attcaacata tttaagtaca
240gataatgaaa aagataatta tttaaaggga gttacaaaat tatttgagag
aatttattca 300actgatcttg gaagaatgtt gttaacatca atagtaaggg
gaataccatt ttggggtgga 360agtacaatag atacagaatt aaaagttatt
gatactaatt gtattaatgt gatacaacca 420gatggtagtt atagatcaga
agaacttaat ctagtaataa taggaccctc agctgatatt 480atacagtttg
aatgtaaaag ctttggacat gaagttttga atcttacgcg aaatggttat
540ggctctactc aatacattag atttagccca gattttacat ttggttttga
ggagtcactt 600gaagttgata caaatcctct tttaggtgca ggcaaatttg
ctacagatcc agcagtaaca 660ttagcacatg aacttataca tgctggacat
agattatatg gaatagcaat taatccaaat 720agggttttta aagtaaatac
taatgcctat tatgaaatga gtgggttaga agtaagcttt 780gaggaactta
gaacatttgg gggacatgat gcaaagttta tagatagttt acaggaaaac
840gaatttcgtc tatattatta taataagttt aaagatatag caagtacact
taataaagct 900aaatcaatag taggtactac tgcttcatta cagtatatga
aaaatgtttt taaagagaaa 960tatctcctat ctgaagatac atctggaaaa
ttttcggtag ataaattaaa atttgataag 1020ttatagaaaa tgttaacaga
gatttagaca gaggataatt ttgttaagtt ttttaaagta 1080cttaacagaa
aaacatattt gaattttgat aaagccgtat ttaagataaa tatagtacct
1140aaggtaaatt acacaatata tgatggattt aatttaagaa atacaaattt
agcagcaaac 1200tttaatggtc aaaatacaga aattaataat atgaatttta
ctaaactaaa aaattttact 1260ggattgtttg aattttataa gttgctatgt
gtaagaggga taataacttc taaaactaaa 1320tcattagata aaggatacaa
taaggcatta aatgatttat gtatcaaagt taataattgg 1380gacttgtttt
ttagtccttc agaagataat tttactaatg atctaaataa aggagaagaa
1440attacatctg atactaatat agaagcagca gaagaaaata ttagtttaga
tttaatacaa 1500caatattatt taacctttaa ttttgataat gaacctgaaa
atatttcaat agaaaatctt 1560tcaagtgaca ttataggcca attagaactt
atgcctaata tagaaagatt tcctaatgga 1620aaaaagtatg agttagataa
atatactatg ttccattatc ttcgtgctca agaatttgaa 1680catggtaaat
ctaggattgc tttaacaaat tctgttaacg aagcattatt aaatcctagt
1740cgtgtttata catttttttc ttcagactat gtaaagaaag ttaataaagc
tacggaggca 1800gctatgtttt taggctgggt agaacaatta gtatatgatt
ttaccgatga aactagcgaa 1860gtaagtacta cggataaaat tgcggatata
actataatta ttccatatat aggacctgct 1920ttaaatatag gtaatatgtt
atataaagat gattttgtag gtgctttaat attttcagga 1980gctgttattc
tgttagaatt tataccagag attgcaatac ctgtattagg tacttttgca
2040cttgtatcat atattgcgaa taaggttcta accgttcaaa caatagataa
tgctttaagt 2100aaaagaaatg aaaaatggga tgaggtctat aaatatatag
taacaaattg gttagcaaag 2160gttaatacac agattgatct aataagaaaa
aaaatgaaag aagctttaga aaatcaagca 2220gaagcaacaa aggctataat
aaactatcag tataatcaat atactgagga agagaaaaat 2280aatattaatt
ttaatattga tgatttaagt tcgaaactta atgagtctat aaataaagct
2340atgattaata taaataaatt tttgaatcaa tgctctgttt catatttaat
gaattctatg 2400atcccttatg gtgttaaacg gttagaagat tttgatgcta
gtcttaaaga tgcattatta 2460aagtatatat atgataatag aggaacttta
attggtcaag tagatagatt aaaagataaa 2520gttaataata cacttagtac
agatatacct tttcagcttt ccaaataggt agataatcaa 2580agattattat
ctacatttac tgaatatatt aagaatatta ttaatacttc tatattgaat
2640ttaagatatg aaagtaatca tttaatagac ttatctaggt atgcatcaaa
aataaatatt 2700ggtagtaaag taaattttga tccaatagat aaaaatcaaa
ttcaattatt taatttagaa 2760agtagtaaaa ttgaggtaat tttaaaaaat
gctattgtat ataatagtat gtatgaaaat 2820tttagtacta gcttttggat
aagaattcct aagtatttta acagtataag tctaaataat 2880gaatatacaa
taataaattg tatggaaaat aattcaggat ggaaagtatc acttaattat
2940ggtgaaataa tctggacttt acaggatact caggaaataa aacaaagagt
agtttttaaa 3000tagagtcaaa tgattaatat atcagattat ataaacagat
ggatttttgt aactatcact 3060aataatagat taaataactc taaaatttat
ataaatggaa gattaataga tgaaaaacca 3120atttcaaatt taggtaatat
tcatgctagt aataatataa tgtttaaatt agatggttgt 3180agagatacac
atagatatat ttggataaaa tattttaatc tttttgataa ggaattaaat
3240gaaaaagaaa tcaaagattt atatgataat caatcaaatt caggtatttt
aaaagacttt 3300tggggtgatt atttacaata tgataaacca tagtatatgt
taaatttata tgatccaaat 3360aaatatgtcg atgtaaataa tgtaggtatt
agaggttata tgtatcttaa agggcctaga 3420ggtagcgtaa tgactacaaa
catttattta aattcaagtt tgtatagggg gacaaaattt 3480attataaaaa
aatatgcttc tggaaataaa gataatattg ttagaaataa tgatcgtgta
3540tatattaatg tagtagttaa aaataaagaa tataggttag ctactaatgc
atcacaggca 3600ggcgtagaaa aaatactaag tgcattagaa atacctgatg
taggaaatct aagtcaagta 3660gtagtaatga agtcaaaaaa tgatcaagga
ataacaaata aatgcaaaat gaatttacaa 3720gataataatg ggaatgatat
aggctttata ggatttcatc agtttaataa tatagctaaa 3780ctagtagcaa
gtaattggta taatagacaa atagaaagat ctagtaggac tttgggttgc
3840tcatgggaat ttattcctgt agatgatgga tggggagaaa ggccactgta a
389123876DNAbotulinum toxin 2atgccagtta caataaataa ttttaattat
aatgatccta ttgataatga caatattatt 60atgatggaac ctccatttgc aaggggtacg
gggagatatt ataaagcttt taaaatcaca 120gatcgtattt ggataatacc
cgaaagatat acttttggat ataaacctga ggattttaat 180aaaagttccg
gtatttttaa tagagatgtt tgtgaatatt atgatccaga ttacttaaat
240accaatgata aaaagaatat atttttccaa acattgatca agttatttaa
tagaatcaaa 300tcaaaaccat tgggtgaaaa gttattagag atgattataa
atggtatacc ttatcttgga 360gatagacgtg ttccactcga agagtttaac
acaaacattg ctagtgtaac tgttaataaa 420ttaattagta atccaggaga
agtggagcga aaaaaaggta ttttcgcaaa tttaataata 480tttggacctg
ggccagtttt aaatgaaaat gagactatag atataggtat acaaaatcat
540tttgcatcaa gggaaggctt tgggggtata atgcaaatga aattttgtcc
agaatatgta 600agcgtattta ataatgttca agaaaacaaa ggcgcaagta
tatttaatag acgtggatat 660ttttcagatc cagccttgat attaatgcat
gaacttatac atgttttgca tggattatat 720ggcattaaag tagatgattt
accaattgta ccaaatgaaa aaaaattttt tatgcaatct 780acagatacta
tacaggcaga agaactatat acatttggag gacaagatcc cagcatcata
840tctccttcta cagataaaag tatctatgat aaagttttgc aaaattttag
ggggatagtt 900gatagactta acaaggtttt agtttgcata tcagatccta
acattaacat taatatatat 960aaaaataaat ttaaagataa atataaattc
gttgaagatt ctgaaggaaa atatagtata 1020gatgtagaaa gtttcaataa
attatataaa agcttaatgt taggttttac agaaattaat 1080atagcagaaa
attataaaat aaaaactaga gcttcttatt ttagtgattc cttaccacca
1140gtaaaaataa aaaatttatt agataatgaa atctatacta tagaggaagg
gtttaatata 1200tctgataaaa atatgggaaa agaatatagg ggtcagaata
aagctataaa taaacaagct 1260tatgaagaaa tcagcaagga gcatttggct
gtatataaga tacaaatgtg taaaagtgtt 1320aaagttccag gaatatgtat
tgatgtcgat aatgaaaatt tgttctttat agctgataaa 1380aatagttttt
cagatgattt atctaaaaat gaaagagtag aatataatac acagaataat
1440tatataggaa atgactttcc tataaatgaa ttaattttag atactgattt
aataagtaaa 1500atagaattac caagtgaaaa tacagaatca cttactgatt
ttaatgtaga tgttccagta 1560tatgaaaaac aacccgctat aaaaaaagtt
tttacagatg aaaataccat ctttcaatat 1620ttatactctc agacatttcc
tctaaatata agagatataa gtttaacatc ttcatttgat 1680gatgcattat
tagtttctag caaagtttat tcattttttt ctatggatta tattaaaact
1740gctaataaag tagtagaagc aggattattt gcaggttggg tgaaacagat
agtagatgat 1800tttgtaatcg aagctaataa aagcagtact atggataaaa
ttgcagatat atctctaatt 1860gttccttata taggattagc tttaaatgta
ggagatgaaa cagctaaagg aaattttgaa 1920agtgcttttg agattgcagg
atccagtatt ttactagaat ttataccaga acttttaata 1980cctgtagttg
gagtcttttt attagaatca tatattgaca ataaaaataa aattattaaa
2040acaatagata atgctttaac taaaagagtg gaaaaatgga ttgatatgta
gggattaata 2100gtagcgcaat ggctctcaac agttaatact caattttata
caataaaaga gggaatgtat 2160aaggctttaa attatcaagc acaagcattg
gaagaaataa taaaatacaa atataatata 2220tattctgaag aggaaaagtc
aaatattaac atcaatttta atgatataaa ttctaaactt 2280aatgatggta
ttaaccaagc tatggataat ataaatgatt ttataaatga atgttctgta
2340tcatatttaa tgaaaaaaat gattccatta gctgtaaaaa aattactaga
ctttgataat 2400actctcaaaa aaaatttatt aaattatata gatgaaaata
aattatattt aattggaagt 2460gtagaagatg aaaaatcaaa agtagataaa
tacttgaaaa ccattatacc atttgatctt 2520tcaacgtatt ctaatattga
aatactaata aaaatattta ataaatataa tagcgaaatt 2580ttaaataata
ttatcttaaa tttaagatat agagataata atttaataga tttatcagga
2640tatggagcaa aggtagaggt atatgatggg gtcaagctta atgataaaaa
tcaatttaaa 2700ttaactagtt cagcagatag taagattaga gtcactcaaa
atcagaatat tatatttaat 2760agtatgttcc ttgattttag cgttagcttt
tggataagga tacctaaata taggaatgat 2820gatatacaaa attatattca
taatgaatat acgataatta attgtatgaa aaataattca 2880ggctggaaaa
tatctattag gggtaatagg ataatatgga ccttaattga tataaatgga
2940aaaaccaaat cagtattttt tgaatataac ataagagaag atatatcaga
gtatataaat 3000agatggtttt ttgtaactat tactaataat ttggataatg
ctaaaattta tattaatggc 3060acgttagaat caaatatgga tattaaagat
ataggagaag ttattgttaa tggtgaaata 3120acatttaaat tagatggtga
tgtagataga acacaattta tttggatgaa atattttagt 3180atttttaata
cgcaattaaa tcaatcaaat attaaagaga tatataaaat tcaatcatat
3240agcgaatagt taaaagattt ttggggaaat cctttaatgt ataataaaga
atattatatg 3300tttaatgcgg ggaataaaaa ttcatatatt aaactagtga
aagattcatc tgtaggtgaa 3360atattaatac gtagcaaata taatcagaat
tccaattata taaattatag aaatttatat 3420attggagaaa aatttattat
aagaagagag tcaaattctc aatctataaa tgatgatata 3480gttagaaaag
aagattatat acatctagat ttggtacttc accatgaaga gtggagagta
3540tatgcctata aatattttaa ggaacaggaa gaaaaattgt ttttatctat
tataagtgat 3600tctaatgaat tttataagac tatagaaata aaagaatatg
atgaacagcc atcatatagt 3660tgtcagttgc tttttaaaaa agatgaagaa
agtactgatg atataggatt gattggtatt 3720catcgtttct aggaatctgg
agttttacgt aaaaagtata aagattattt ttgtataagt 3780aaatggtagt
taaaagaggt aaaaaggaaa ccatataagt caaatttggg atgtaattgg
3840cagtttattc ctaaagatga agggtggact gaataa 387633876DNAbotulinum
toxin 3atgccagtta caataaataa ttttaattat aatgatccta ttgataataa
taatattatt 60atgatggagc ctccatttgc gagaggtacg gggagatatt ataaagcttt
taaaatcaca 120gatcgtattt ggataatacc ggaaagatat acttttggat
ataaacctga ggattttaat 180aaaagttccg gtatttttaa tagagatgtt
tgtgaatatt atgatccaga ttagttaaat 240actaatgata aaaagaatat
atttttacaa acaatgatca agttatttaa tagaatcaaa 300tcaaaaccat
tgggtgaaaa gttattagag atgattataa atggtatacc ttatcttgga
360gatagacgtg ttccactcga agagtttaac acaaacattg ctagtgtaac
tgttaataaa 420ttaatcagta atccaggaga agtggagcga aaaaaaggta
ttttcgcaaa tttaataata 480tttggacctg ggccagtttt aaatgaaaat
gagactatag atataggtat acaaaatcat 540tttgcatcaa gggaaggctt
cgggggtata atgcaaatga agttttgccc agaatatgta 600agcgtattta
ataatgttca agaaaacaaa ggcgcaagta tatttaatag acgtggatat
660ttttcagatc cagccttgat attaatgcat gaacttatac atgttttaca
tggattatat 720ggcattaaag tagatgattt accaattgta ccaaatgaaa
aaaaattttt tatgcaatct 780acagatgcta tacaggcaga agaactatat
acatttggag gacaagatcc cagcatcata 840actccttcta cggataaaag
tatctatgat aaagttttgc aaaattttag agggatagtt 900gatagactta
acaaggtttt agtttgcata tcagatccta acattaatat taatatatat
960aaaaataaat ttaaagataa atataaattc gttgaagatt ctgagggaaa
atatagtata 1020gatgtagaaa gttttgataa attatataaa agcttaatgt
ttggttttac agaaactaat 1080atagcagaaa attataaaat aaaaactaga
gcttcttatt ttagtgattc cttaccacca 1140gtaaaaataa aaaatttatt
agataatgaa atctatacta tagaggaagg gtttaatata 1200tctgataaag
atatggaaaa agaatataga ggtcagaata aagctataaa taaacaagct
1260tatgaagaaa ttagcaagga gcatttggct gtatataaga tacaaatgtg
taaaagtgtt 1320aaagctccag gaatatgtat tgatgttgat aatgaagatt
tgttctttat agctgataaa 1380aatagttttt cagatgattt atctaaaaac
gaaagaatag aatataatac acagagtaat 1440tatatagaaa atgacttccc
tataaatgaa ttaattttag atactgattt aataagtaaa 1500atagaattac
caagtgaaaa tacagaatca cttactgatt ttaatgtaga tgttccagta
1560tatgaaaaac aacccgctat aaaaaaaatt tttacagatg aaaataccat
ctttcaatat 1620ttatagtctc agacatttct cttagatata agagatataa
gtttaacatc ttcatttgat 1680gatgcattat tattttctaa caaagtttat
tcattttttt ctatggatta tattaaaact 1740gctaataaag tggtagaagc
aggattattt gcaggttggg tgaaacagat agtaaatgat 1800tttgtaatcg
aagctaataa aagcaatact atggataaaa ttgcagatat atctctaatt
1860gttccttata taggattagc tttaaatgta ggaaatgaaa cagctaaagg
aaattttgaa 1920aatgcttttg agattgcagg agccagtatt ctactagaat
ttataccaga acttttaata 1980cctgtagttg gagccttttt attagaatca
tatattgaca ataaaaataa aattattaaa 2040acaatagata atgctttaac
taaaagaaat gaaaaatgga gtgatatgta gggattaata 2100gtagcgcaat
ggctctcaac agttaatact caattttata caataaaaga gggaatgtat
2160aaggctttaa attatcaagc acaagcattg gaagaaataa taaaatacag
atataatata 2220tattctgaaa aagaaaagtc aaatattaac atcgatttta
atgatataaa ttctaaactt 2280aatgagggta ttaaccaagc tatagataat
ataaataatt ttataaatgg atgttctgta 2340tcatatttaa tgaaaaaaat
gattccatta gctgtagaaa aattactaga ctttgataat 2400actctcaaaa
aaaatttgtt aaattatata gatgaaaata aattatattt gattggaagt
2460gcagaatatg aaaaatcaaa agtaaataaa tagttgaaaa ccattatgcc
gtttgatctt 2520tcaatatata ccaatgatac aatactaata gaaatgttta
ataaatataa tagcgaaatt 2580ttaaataata ttatcttaaa tttaagatat
aaggataata atttaataga tttatcagga 2640tatggggcaa aggtagaggt
atatgatgga gtcgagctta atgataaaaa tcaatttaaa 2700ttaactagtt
cagcaaatag taagattaga gtgactcaaa atcagaatat catatttaat
2760agtgtgttcc ttgattttag cgttagcttt tggataagaa tacctaaata
taagaatgat 2820ggtatacaaa attatattca taatgaatat acaataatta
attgtatgaa aaataattcg 2880ggctggaaaa tatctattag gggtaatagg
ataatatgga ctttaattga tataaatgga 2940aaaaccaaat cggtattttt
tgaatataac ataagagaag atatatcaga gtatataaat 3000agatggtttt
ttgtaactat tactaataat ttgaataacg ctaaaattta tattaatggt
3060aagctagaat caaatacaga tattaaagat ataagagaag ttattgctaa
tggtgaaata 3120atatttaaat tagatggtga tatagataga acacaattta
tttggatgaa atatttcagt 3180atttttaata cggaattaag tcaatcaaat
attgaagaaa gatataaaat tcaatcatat 3240agcgaatatt taaaagattt
ttggggaaat cctttaatgt agaataaaga atattatatg 3300tttaatgcgg
ggaataaaaa ttcatatatt aaactaaaga aagattcacc tgtaggtgaa
3360attttaacac gtagcaaata taatcaaaat tctaaatata taaattatag
agatttatat 3420attggagaaa aatttattat aagaagaaag tcaaattctc
aatctataaa tgatgatata 3480gttagaaaag aagattatat atatctagat
ttttttaatt taaatcaaga gtggagagta 3540tatacctata aatattttaa
gaaagaggaa gaaaaattgt ttttagctcc tataagtgat 3600tctgatgagt
tttagaatac tatacaaata aaagaatatg atgaacagcc aacatatast
3660tgtcagttgc tttttaaaaa agatgaagaa agtactgatg agataggatt
gattggtatt 3720catcgtttct aggaatctgg aattgtattt gaagagtata
aagattattt ttgtataagt 3780aaatggtagt taaaagaggt aaaaaggaaa
ccatataatt taaaattggg atgtaattgg 3840cagtttattc ctaaagatga
agggtggact gaataa 387643876DNAbotulinum toxin 4atgccaataa
caattaacaa ctttaattat tcagatcctg ttgataataa aaatatttta 60tatttagata
ctcatttaaa tacactagct aatgagcctg aaaaagcctt tcgcattaca
120ggaaatatat gggtaatacc tgatagattt tcaagaaatt ctaatccaaa
tttaaataaa 180cctcctcgag ttacaagccc taaaagtggt tattatgatc
ctaattattt gagtactgat 240tctgacaaag atacattttt aaaagaaatt
ataaagttat ttaaaagaat taattctaga 300gaaataggag aagaattaat
atatagactt tcgacagata taccctttcc tgggaataac 360aatactccaa
ttaatacttt tgattttgat gtagatttta acagtgttga tgttaaaact
420agacaaggta acaactgggt taaaactggt agcataaatc ctagtgttat
aataactgga 480cctagagaaa acattataga tccagaaact tctacgttta
aattaactaa caatactttt 540gcggcacaag aaggatttgg tgctttatca
ataatttcaa tatcacctag atttatgcta 600acatatagta atgcaactaa
tgatgtagga gagggtagat tttctaagtc tgaattttgc 660atggatccaa
tactaatttt aatgcatgaa cttaatcatg caatgcataa tttatatgga
720atagctatac caaatgatca aacaatttca tctgtaacta gtaatatttt
ttattctcaa 780tataatgtga aattagagta tgcagaaata tatgcatttg
gaggtccaac tatagacctt 840attcctaaaa gtgcaaggaa atattttgag
gaaaaggcat tggattatta tagatctata 900gctaaaagac ttaatagtat
aactactgca aatccttcaa gctttaataa atatataggg 960gaatataaac
agaaacttat tagaaagtat agattcgtag tagaatcttc aggtgaagtt
1020acagtaaatc gtaataagtt tgttgagtta tataatgaac ttacacaaat
atttacagaa 1080tttaactagg ctaaaatata taatgtacaa aataggaaaa
tatatctttc aaatgtatat 1140actccggtta cggcgaatat attagacgat
aatgtttatg atatacaaaa tggatttaat 1200atacctaaaa gtaatttaaa
tgtactattt atgggtcaaa atttatctcg aaatccagca 1260ttaagaaaag
tcaatcctga aaatatgctt tatttattta caaaattttg tcataaagca
1320atagatggta gatcattata taataaaaca ttagattgta gagagctttt
agttaaaaat 1380actgacttac cctttatagg tgatattagt gatgttaaaa
ctgatatatt tttaagaaaa 1440gatattaatg aagaaactga agttatatac
tatccggaca atgtttcagt agatcaagtt 1500attctcagta agaatacctc
agaacatgga caactagatt tattataccc tagtattgac 1560agtgagagtg
aaatattacc aggggagaat caagtctttt atgataatag aactcaaaat
1620gttgattatt tgaattctta ttattaccta gaatctcaaa aactaagtga
taatgttgaa 1680gattttactt ttacgagatc aattgaggag gctttggata
atagtgcaaa agtatatact 1740tactttccta cactagctaa taaagtaaat
gcgggtgttc aaggtggttt atttttaatg 1800tgggcaaatg atgtagttga
agattttact acaaatattc taagaaaaga tacattagat 1860aaaatatcag
atgtatcagc tattattccc tatataggac ccgcattaaa tataagtaat
1920tctgtaagaa gaggaaattt tactgaagca tttgcagtta ctggtgtaac
tattttatta 1980gaagcatttc ctgaatttac aatacctgca cttggtgcat
ttgtgattta tagtaaggtt 2040caagaaagaa acgagattat taaaactata
gataattgtt tagaacaaag gattaagaga 2100tggaaagatt catatgaatg
gatgatggga acgtggttat ccaggattat tactcaattt 2160aataatataa
gttatcaaat gtatgattct ttaaattatc aggcaggtgc aatcaaagct
2220aaaatagatt tagaatataa aaaatattca ggaagtgata aagaaaatat
aaaaagtcaa 2280gttgaaaatt taaaaaatag tttagatgta aaaatttcgg
aagcaatgaa taatataaat 2340aaatttatac gagaatgttc cgtaacatat
ttatttaaaa atatgttacc taaagtaatt 2400gatgaattaa atgagtttga
tcgaaatact aaagcaaaat taattaatct tatagatagt 2460cataatatta
ttctagttgg tgaagtagat aaattaaaag caaaagtaaa taatagcttt
2520caaaatacaa taccctttaa tattttttca tatactaata attctttatt
aaaagatata 2580attaatgaat atttcaataa tattaatgat tcaaaaattt
tgagcctaca aaacagaaaa 2640aatactttag tggatacatc aggatataat
gcagaagtga gtgaagaagg cgatgttcag 2700cttaatccaa tatttccatt
tgactttaaa ttaggtagtt caggggagga tagaggtaaa 2760gttatagtaa
cccagaatga aaatattgta tataattcta tgtatgaaag ttttagcatt
2820agtttttgga ttagaataaa taaatgggta agtaatttac ctggatatac
tataattgat 2880agtgttaaaa ataactcagg ttggagtata ggtattatta
gtaatttttt agtatttact 2940ttaaaacaaa atgaagatag tgaacaaagt
ataaatttta gttatgatat atcaaataat 3000gctcctggat agaataaatg
gttttttgta actgttacta acaatatgat gggaaatatg 3060aagatttata
taaatggaaa attaatagat actataaaag ttaaagaact aactggaatt
3120aattttagca aaactataac atttgaaata aataaaattc cagataccgg
tttgattact 3180tcagattctg ataacatcaa tatgtggata agagattttt
atatatttgc taaagaatta 3240gatggtaaag atattaatat
attatttaat agcttgcaat atactaatgt tgtaaaagat 3300tattggggaa
atgatttaag atataataaa gaatattata tggttaatat agattattta
3360aatagatata tgtatgcgaa ctcacgacaa attgttttta atacacgtag
aaataataat 3420gacttcaatg aaggatataa aattataata aaaagaatca
gaggaaatac aaatgatact 3480agagtacgag gaggagatat tttatatttt
gatatgacaa ttaataacaa agcatataat 3540ttgtttatga agaatgaaac
tatgtatgca gataatcata gtactgaaga tatatatgct 3600ataggtttaa
gagaacaaac aaaggatata aatgataata ttatatttca aatacaacca
3660atgaataata cttattatta ggcatctcaa atatttaaat caaattttaa
tggagaaaat 3720atttctggaa tatgttcaat aggtacttat cgttttagac
ttggaggtga ttggtataga 3780cacaattatt tggtgcctac tgtgaagcaa
ggaaattatg cttcattatt agaatcaaca 3840tcaactcatt ggggttttgt
acctgtaagt gaataa 387653831DNAbotulinum toxin 5atgacatggc
cagtaaaaga ttttaattat agtgatcctg ttaatgacaa tgatatatta 60tatttaagaa
taccacaaaa taagttaatt actacacctg taaaagcttt tatgattact
120caaaatattt gggtaatacc agaaagattt tcatcagata ctaatccaag
tttaagtaaa 180ccgcccagac ctacttcaaa gtatcaaagt tattatgatc
ctagttattt atctactgat 240gaacaaaaag atacattttt aaaagggatt
ataaaattat ttaaaagaat taatgaaaga 300gatataggaa aaaaattaat
aaattattta gtagttggtt caccttttat gggagattca 360agtacgcctg
aagatacatt tgattttaca cgtcatacta ctaatattgc agttgaaaag
420tttgaaaatg gtagttggaa agtaacaaat attataacac caagtgtatt
gatatttgga 480ccacttccta atatattaga ctatacagca tcccttacat
tgcaaggaca acaatcaaat 540ccatcatttg aagggtttgg aacattatct
atactaaaag tagcacctga atttttgtta 600acatttagtg atgtaacatc
taatcaaagt tcagctgtat taggcaaatc tatattttgt 660atggatccag
taatagcttt aatgcatgag ttaacacatt ctttgcatca attatatgga
720ataaatatac catctgataa aaggattcgt ccacaagtta gcgagggatt
tttctctcaa 780gatggaccca acgtacaatt tgaggaatta tatacatttg
gaggattaga tgttgaaata 840atacctcaaa ttgaaagatc acaattaaga
gaaaaagcat taggtcacta taaagatata 900gcgaaaagac ttaataatat
taataaaact attccttcta gttggattag taatatagat 960aaatataaaa
aaatattttc tgaaaagtat aattttgata aagataatac aggaaatttt
1020gttgtaaata ttgataaatt caatagctta tattcagact tgactaatgt
tatgtcagaa 1080gttgtttatt cttcgcaata taatgttaaa aacaggactc
attatttttc aaggcattat 1140ctacctgtat ttgcaaatat attagatgat
aatatttata ctataagaga tggttttaat 1200ttaacaaata aaggttttaa
tatagaaaat tcgggtcaga atatagaaag gaatcctgca 1260ctacaaaagc
ttagttcaga aagtgtagta gatttattta caaaagtatg tttaagatta
1320acaaaaaata gtagagatga ttcaacatgt attaaagtta aaaataatag
attaccttat 1380gtagctgata aagatagcat ttcacaagaa atatttgaaa
ataaaattat tacagatgag 1440actaatgtac aaaattattc agataatttt
tcattagatg aatctatttt agatgggcaa 1500gttcctatta atcctgaaat
agtagatcca ctattaccca atgttaatat ggaaccttta 1560aatcttccag
gtgaagaaat agtattttat gatgatatta ctaaatatgt tgattattta
1620aattcttatt attatttgga atctcaaaaa ttaagtaata atgttgaaaa
tattactctt 1680acaacttcag ttgaagaagc attaggttat agcaataaga
tatagacatt tttacctagc 1740ttagctgaaa aagtgaataa aggtgttcaa
gcaggtttat tcttaaattg ggcgaatgaa 1800gtagttgagg attttactac
aaatattatg aagaaagata cattggataa aatatcagat 1860gtatcagtaa
taattccata tataggacct gccttaaata taggaaattc agcattaagg
1920ggaaatttta agcaagcatt tgcaacagct ggtgtagctt ttttattaga
gggatttcca 1980gagtttacta tacctgcact cggtgtattt accttttata
gttctattca agaaagagag 2040aaaattatta aaactataga aaattgtttg
gaacaaagag ttaagagatg gaaagattca 2100tatcaatgga tggtatcaaa
ttggttgtca agaattacta ctcaatttaa tcatataaat 2160tatcaaatgt
atgattcttt aagttatcag gcagatgcaa tcaaagctaa aatagattta
2220gaatataaaa aatagtcagg aagtgataaa gaaaatataa aaagtcaagt
tgaaaattta 2280aaaaatagtt tagatgtaaa aatttcggaa gcaatgaata
atataaataa atttatacga 2340gaatgttctg taacatagtt atttaaaaat
atgctcccta aagtaattga cgaattaaat 2400aagtttgatt taagaactaa
aacagaatta attaatctta tagatagtca taatattatt 2460ctagttggtg
aagtagatag attaaaagca aaagtaaatg agagttttga aaatacaatg
2520ccttttaata ttttttcata tactaataat tctttattaa aagatataat
taatgaatat 2580ttcaatagta ttaatgattc aaaaattttg agcttacaaa
acaaaaaaaa tgctttagtg 2640gatacatcag gatataatgc agaagtgagg
gtaggagata atgttcaact taatacgata 2700tatacaaatg actttaaatt
aagtagttca ggagataaaa ttatagtaaa tttaaataat 2760aatattttat
atagcgctat ttatgagaac tctagtgtta gtttttggat taagatatct
2820aaagatttaa ctaattctca taatgaatat acaataatta acagtataga
acaaaattct 2880gggtggaaat tatgtattag gaatggcaat atagaatgga
ttttacaaga tgttaataga 2940aagtataaaa gtttaatttt tgattatagt
gaatcattaa gtcatacagg atatacaaat 3000aaatggtttt ttgttactat
aactaataat ataatggggt atatgaaact ttatataaat 3060ggagaattaa
agcagagtca aaaaattgaa gatttagatg aggttaagtt agataaaacc
3120atagtatttg gaatagatga gaatatagat gagaatcaga tgctttggat
tagagatttt 3180aatatttttt ctaaagaatt aagtaatgaa gatattaata
ttgtatatga gggacaaata 3240ttaagaaatg ttattaaaga ttattgggga
aatcctttga agtttgatac agaatattat 3300attattaatg ataattatat
agataggtat attgcacctg aaagtaatgt acttgtactt 3360gttcggtatc
cagatagatc taaattatat actggaaatc ctattactat taaatcagta
3420tctgataaga atccttatag tagaatttta aatggagata atataattct
tcatatgtta 3480tataatagta ggaaatatat gataataaga gatactgata
caatatatgc aacacaagga 3540ggagagtgtt cacaaaattg tgtatatgca
ttaaaattac agagtaattt aggtaattat 3600ggtataggta tatttagtat
aaaaaatatt gtatctaaaa ataaatattg tagtcaaatt 3660ttctctagtt
ttagggaaaa tacaatgctt ctagcagata tatataaacc ttggagattt
3720tcttttaaaa atgcatagac gccagttgca gtaactaatt atgaaacaaa
actattatca 3780acttcatctt tttggaaatt tatttctagg gatccaggat
gggtagagta a 383163753DNAbotulinum toxin 6atgccaacaa ttaatagttt
taattataat gatcctgtta ataatagaac aattttatat 60attaaaccag gcggttgtca
acaattttat aaatcattta atattatgaa aaatatttgg 120ataattccag
agagaaatgt aattggtaca attccccaag attttcttcc gcctacttca
180ttgaaaaatg gagatagtag ttattatgac cctaattatt tacaaagtga
tcaagaaaag 240gataaatttt taaaaatagt cacaaaaata tttaatagaa
taaatgataa tctttcagga 300aggattttat tagaagaact gtcaaaagct
aatccatatt taggaaatga taatactcca 360gatggtgact tcattattaa
tgatgcatca gcagttccaa ttcaattctc aaatggtagc 420caaagcatac
tattacctaa tgttattata atgggagcag agcctgattt atttgaaact
480aacagttcca atatttctct aagaaataat tatatgccaa gcaatcacgg
ttttggatca 540atagctatag taacattctc acctgaatat tcttttagat
ttaaagataa tagtatgaat 600gaatttattc aagatcctgc tcttacatta
atgcatgaat taatacattc attacatgga 660ctatatgggg ctaaagggat
tactacaaag tatactataa cacaaaaaca aaatccccta 720ataacaaata
taagaggtac aaatattgaa gaattcttaa cttttggagg tactgattta
780aacattatta ctagtgctca gtccaatgat atctatacta atcttctagc
tgattataaa 840aaaatagcgt ctaaacttag caaagtacaa gtatctaatc
cactacttaa tccttataaa 900gatgtttttg aagcaaagta tggattagat
aaagatgcta gcggaattta ttcggtaaat 960ataaacaaat ttaatgatat
ttttaaaaaa ttatacagct ttacggaatt tgatttagca 1020actaaatttc
aagttaaatg taggcaaact tatattggac agtataaata cttcaaactt
1080tcaaacttgt taaatgattc tatttataat atatcagaag gctataatat
aaataattta 1140aaggtaaatt ttagaggaca gaatgcaaat ttaaatccta
gaattattac accaattaca 1200ggtagaggac tagtaaaaaa aatcattaga
ttttgtaaaa atattgtttc tgtaaaaggc 1260ataaggaaat caatatgtat
cgaaataaat aatggtgagt tattttttgt ggcttccgag 1320aatagttata
atgatgataa tataaatact cctaaagaaa ttgacgatac agtaacttca
1380aataataatt atgaaaatga tttagatcag gttattttaa attttaatag
tgaatcagca 1440cctggacttt cagatgaaaa attaaattta actatccaaa
atgatgctta tataccaaaa 1500tatgattcta atggaacaag tgatatagaa
caacatgatg ttaatgaact taatgtattt 1560ttctatttag atgcacagaa
agtgcccgaa ggtgaaaata atgtcaatct cacctcttca 1620attgatacag
cattattaga acaacctaaa atatatacat ttttttcatc agaatttatt
1680aataatgtca ataaacctgt gcaagcagca ttatttgtaa gctggataca
acaagtatta 1740gtagatttta ctactgaagc taaccaaaaa agtactgttg
ataaaattgc agatatttct 1800atagttgttc catatatagg tcttgcttta
aatataggaa atgaagcaca aaaaggaaat 1860tttaaagatg cacttgaatt
attaggagca ggtattttat tagaatttga acccgagctt 1920ttaattccta
caattttagt attcacgata aaatcttttt taggttcatc tgataataaa
1980aataaagtta ttaaagcaat aaataatgca ttgaaagaaa gagatgaaaa
atggaaagaa 2040gtatatagtt ttatagtatc gaattggatg actaaaatta
atacacaatt taataaaaga 2100aaagaacaaa tgtatcaagc tttacaaaat
caagtaaatg cacttaaagc aataatagaa 2160tctaagtata atagttatac
tttagaagaa aaaaatgagc ttacaaataa atatgatatt 2220gagcaaatag
aaaatgaact taatcaaaag gtttctatag caatgaataa tatagacagg
2280ttcttaactg aaagttctat atcttattta atgaaattaa taaatgaagt
aaaaattaat 2340aaattaagag aatatgatga aaatgttaaa acgtatttat
tagattatat tataaaacat 2400ggatcaatct tgggagagag tcagcaagaa
ctaaattcta tggtaattga taccctaaat 2460aatagtattc cttttaagct
ttcttcttat acagatgata aaattttaat ttcatatttt 2520aataagttct
ttaagagaat taaaagtagt tctgttttaa atatgagata taaaaatgat
2580aaataggtag atacttcagg atatgattca aatataaata ttaatggaga
tgtatataaa 2640tatccaacta ataaaaatca atttggaata tataatgata
aacttagtga agttaatata 2700tctcaaaatg attacattat atatgataat
aaatataaaa attttagtat tagtttttgg 2760gtaagaattc ctaactatga
taataagata gtaaatgtta ataatgaata cactataata 2820aattgtatga
gggataataa ttcaggatgg aaagtatctc ttaatcataa tgaaataatt
2880tggacattgc aagataattc aggaattaat caaaaattag catttaacta
tggtaacgca 2940aatggtattt ctgattatat aaataagtgg atttttgtaa
ctataactaa tgatagatta 3000ggagattcta aactttatat taatggaaat
ttaatagata aaaaatcaat tttaaattta 3060ggtaatattc atgttagtga
caatatatta tttaaaatag ttaattgtag ttatacaaga 3120tatattggta
ttagatattt taatattttt gataaagaat tagatgaaac agaaattcaa
3180actttatata acaatgaacc taatgcaaat attttaaagg atttttgggg
aaattatttg 3240ctttatgaca aagaatagta tttattaaat gtgttaaaac
caaataactt tattaatagg 3300agaacagatt ctactttaag cattaataat
ataagaagca ctattctttt agctaataga 3360ttatatagtg gaataaaagt
taaaatacaa agagttaata atagtagtac taacgataat 3420cttgttagaa
agaatgatca ggtatatatt aattttgtag ccagcaaaac tcacttactt
3480ccattatatg ctgatacagc taccacaaat aaagagaaaa caataaaaat
atcatcatct 3540ggcaatagat ttaatcaagt agtagttatg aattcagtag
gatgtacaat gaattttaaa 3600aataataatg gaaataatat tgggttgtta
ggtttcaagg cagatactgt agttgctagt 3660acttggtatt atacacatat
gagagataat acaaacagca atggattttt ttggaacttt 3720atttctgaag
aacatggatg gcaagaaaaa taa 375373759DNAbotulinum toxin 7atgccaaaaa
ttaatagttt taattataat gatcctgtta atgatagaac aattttatat 60attaaaccag
gcggttgtca agaattttat aaatcattta atattatgaa aaatatttgg
120ataattccag agagaaatgt aattggtaca accccccaag attttcatcc
gcctacttca 180ttaaaaaatg gagatagtag ttattatgac cctaattatt
tacaaagtga tgaagaaaag 240gatagatttt taaaaatagt cacaaaaata
tttaatagaa taaataataa tctttcagga 300gggattttat tagaagaact
gtcaaaagct aatccatatt tagggaatga taatactcca 360gataatcaat
tccatattgg tgatgcatca gcagttgaga ttaaattctc aaatggtagc
420caagacatac tattacctaa tgttattata atgggagcag agcctgattt
atttgaaact 480aacagttcca atatttctct aagaaataat tatatgccaa
gcaatcacgg ttttggatca 540atagctatag taacattctc acctgaatat
tcttttagat ttaatgataa tagtatgaat 600gaatttattc aagatcctgc
tcttacatta atgcatgaat taatacattc attacatgga 660ctatatgggg
ctaaagggat tactacaaag tatactataa cacaaaaaca aaatccccta
720ataacaaata taagaggtac aaatattgaa gaattcttaa cttttggagg
tactgattta 780aacattatta ctagtgctca gtccaatgat atctatacta
atcttctagc tgattataaa 840aaaatagcgt ctaaacttag caaagtacaa
gtatctaatc cactacttaa tccttataaa 900gatgtttttg aagcaaagta
tggattagat aaagatgcta gcggaattta ttcggtaaat 960ataaacaaat
ttaatgatat ttttaaaaaa ttatagagct ttacggaatt tgatttagca
1020actaaatttc aagttaaatg taggcaaact tatattggac agtataaata
cttcaaactt 1080tcaaacttgt taaatgattc tatttataat atatcagaag
gctataatat aaataattta 1140aaggtaaatt ttagaggaca gaatgcaaat
ttaaatccta gaattattac accaattaca 1200ggtagaggac tagtaaaaaa
aatcattaga ttttgtaaaa atattgtttc tgtaaaaggc 1260ataaggaaat
caatatgtat cgaaataaat aatggtgagt tattttttgt ggcttccgag
1320aatagttata atgatgataa tataaatact cctaaagaaa ttgacgatac
agtaacttca 1380aataataatt atgaaaatga tttagatcag gttattttaa
attttaatag tgaatcagca 1440cctggacttt cagatgaaaa attaaattta
actatccaaa atgatgctta tataccaaaa 1500tatgattcta atggaacaag
tgatatagaa caacatgatg ttaatgaact taatgtattt 1560ttctatttag
atgcacagaa agtgcccgaa ggtgaaaata atgtcaatct cacctcttca
1620attgatacag cattattaga acaacctaaa atatatacat ttttttcatc
agaatttatt 1680aataatgtca ataaacctgt gcaagcagca ttatttgtaa
gctggataca acaagtgtta 1740gtagatttta ctactgaagc taaccaaaaa
agtactgttg ataaaattgc agatatttct 1800atagttgttc catatatagg
tcttgcttta aatataggaa atgaagcaca aaaaggaaat 1860tttaaagatg
cacttgaatt attaggagca ggtattttat tagaatttga acccgagctt
1920ttaattccta caattttagt attcacgata aaatcttttt taggttcatc
tgataataaa 1980aataaagtta ttaaagcaat aaataatgca ttgaaagaaa
gagatgaaaa atggaaagaa 2040gtatatagtt ttatagtatc gaattggatg
actaaaatta atacacaatt taataaaaga 2100aaagaacaaa tgtatcaagc
tttacaaaat caagtaaatg caattaaaac aataatagaa 2160tctaagtata
atagttatac tttagaggaa aaaaatgagc ttacaaataa atatgatatt
2220aagcaaatag aaaatgaact taatcaaaag gtttctatag caatgaataa
tatagacagg 2280ttcttaactg aaagttctat atcctattta atgaaattaa
taaatgaagt aaaaattaat 2340aaattaagag aatatgatga gaatgtcaaa
acgtatttat tgaattatat tatacaacat 2400ggatcaatct tgggagagag
tcagcaagaa ctaaattcta tggtaactga taccctaaat 2460aatagtattc
cttttaagct ttcttcttat acagatgata aaattttaat ttcatatttt
2520aataaattct ttaagagaat taaaagtagt tcagttttaa atatgagata
taaaaatgat 2580aaatacgtag atacttcagg atatgattca aatataaata
ttaatggaga tgtatataaa 2640tatccaacta ataaaaatca atttggaata
tataatgata aacttagtga agttaatata 2700tctcaaaatg attagattat
atatgataat aaatataaaa attttagtat tagtttttgg 2760gtaagaattc
ctaactatga taataagata gtaaatgtta ataatgaata gactataata
2820aattgtatga gagataataa ttcaggatgg aaagtatctc ttaatcataa
tgaaataatt 2880tggacattgc aagataatgc aggaattaat caaaaattag
catttaacta tggtaacgca 2940aatggtattt ctgattatat aaataagtgg
atttttgtaa ctataactaa tgatagatta 3000ggagattcta aactttatat
taatggaaat ttaatagatc aaaaatcaat tttaaattta 3060ggtaatattc
atgttagtga caatatatta tttaaaatag ttaattgtag ttatacaaga
3120tatattggta ttagatattt taatattttt gataaagaat tagatgaaac
agaaattcaa 3180actttatata gcaatgaacc taatacaaat attttgaagg
atttttgggg aaattatttg 3240ctttatgaca aagaatacta tttattaaat
gtgttaaaac caaataactt tattgatagg 3300agaaaagatt ctactttaag
cattaataat ataagaagca ctattctttt agctaataga 3360ttatatagtg
gaataaaagt taaaatacaa agagttaata atagtagtac taacgataat
3420cttgttagaa agaatgatca ggtatatatt aattttgtag ccagcaaaac
tcacttattt 3480ccattatatg ctgatacagc taccacaaat aaagagaaaa
caataaaaat atcatcatct 3540ggcaatagat ttaatcaagt agtagttatg
aattcagtag gaaataattg tacaatgaat 3600tttaaaaata ataatggaaa
taatattggg ttgttaggtt tcaaggcaga tactgtagtt 3660gctagtactt
ggtattatac acatatgaga gatcatacaa acagcaatgg atgtttttgg
3720aactttattt ctgaagaaca tggatggcaa gaaaaataa
375983825DNAbotulinum toxin 8atgccagttg caataaatag ttttaattat
aatgaccctg ttaatgatga tacaatttta 60tagatgcaga taccatatga agaaaaaagt
aaaaaatatt ataaagcttt tgagattatg 120cgtaatgttt ggataattcc
tgagagaaat acaataggaa cgaatcctag tgattttgat 180ccaccggctt
cattaaagaa cggaagcagt gcttattatg atcctaatta tttaaccact
240gatgctgaaa aagatagata tttaaaaaca acgataaaat tatttaagag
aattaatagt 300aatcctgcag ggaaagtttt gttacaagaa atatcatatg
ctaaaccata tttaggaaat 360gaccacacgc caattgatga attctctcca
gttactagaa ctacaagtgt taatataaaa 420ttatcaacta atgttgaaag
ttcaatgtta ttgaatcttc ttgtattggg agcaggacct 480gatatatttg
aaagttgttg ttaccccgtt agaaaactaa tagatccaga tgtagtttat
540gatccaagta attatggttt tggatcaatt aatatcgtga cattttcacc
tgagtatgaa 600tatactttta atgatattag tggagggcat aatagtagta
cagaatcatt tattgcagat 660cctgcaattt cactagctca tgaattgata
catgcactgc atggattata cggggctagg 720ggagttactt atgaagagac
tatagaagta aagcaagcac ctcttatgat agccgaaaaa 780cccataaggc
tagaagaatt tttaaccttt ggaggtcagg atttaaatat tattactagt
840gctatgaagg aaaaaatata taacaatctt ttagctaact atgaaaaaat
agctactaga 900cttagtgaag ttaatagtgc tcctcctgaa tatgatatta
atgaatataa agattatttt 960caatggaagt atgggctaga taaaaatgct
gatggaagtt atactgtaaa tgaaaataaa 1020tttaatgaaa tttataaaaa
attatatagt tttacagaga gtgacttagc aaataaattt 1080aaagtaaaat
gtagaaatac ttattttatt aaatatgaat ttttaaaagt tccaaatttg
1140ttagatgatg atatttatac tgtatcagag gggtttaata taggtaattt
agcagtaaac 1200aatcgcggac aaagtataaa gttaaatcct aaaattattg
attccattcc agataaaggt 1260ctagtagaaa agatcgttaa attttgtaag
agcgttattc ctagaaaagg tacaaaggcg 1320ccaccgcgac tatgcattag
agtaaataat agtgagttat tttttgtagc ttcagaaagt 1380agctataatg
aaaatgatat taatacacct aaagaaattg acgatacaac aaatctaaat
1440aataattata gaaataattt agatgaagtt attttagatt ataatagtca
gacaatacct 1500caaatatcaa atcgaacatt aaatacactt gtacaagaca
atagttatgt gccaagatat 1560gattctaatg gaacaagtga aatagaggaa
tatgatgttg ttgactttaa tgtatttttc 1620tatttacatg cacaaaaagt
gccagaaggt gaaaccaata taagtttaac ttcttcaatt 1680gatacagcat
tattagaaga atccaaagat atattttttt cttcagagtt tatcgatact
1740atcaataaac ctgtaaatgc agcactattt atagattgga taagcaaagt
aataagagat 1800tttaccactg aagctacaca aaaaagtact gttgataaga
ttgcagacat atctttaatt 1860gtaccctatg taggtcttgc tttgaatata
attattgagg cagaaaaagg aaattttgag 1920gaggcatttg aattattagg
agtgggtatt ttattagaat ttgtgccaga acttacaatt 1980cctgtaattt
tagtgtttac gataaaatcc tatatagatt catatgagaa taaaaataaa
2040gcaattaaag caataaataa ttcattaatc gaaagagaag caaagtggaa
agaaatatat 2100agttggatag tatcaaattg gcttactaga attaatactc
aatttaataa aagaaaagag 2160caaatgtatc aggctttaca aaatcaagta
gatgcaataa aaacagcaat agaatataaa 2220tataataatt atacttcaga
tgagaaaaat agacttgaat ctgaatataa tatcaataat 2280atagaagaag
aattgaataa aaaagtttct ttagcaatga aaaatataga aagatttatg
2340acagaaagtt ctatatctta tttaatgaaa ttaataaatg aagccaaagt
tggtaaatta 2400aaaaaatatg ataaccatgt taagagcgat ttattaaact
atattctcga ccatagatca 2460atcttaggag agcagacaaa tgaattaagt
gatttggtga ctagtacttt gaatagtagt 2520attccatttg aactttcttc
atatactaat gataaaattc taattatata ttttaataga 2580ttatataaaa
aaattaaaga tagttctatt ttagatatgc gatatgaaaa taataaattt
2640atagatatct ctggatatgg ttcaaatata agcattaatg gaaacgtata
tatttattca 2700acaaatagaa atcaatttgg aatatataat agtaggctta
gtgaagttaa tatagctcaa 2760aataatgata ttatatagaa tagtagatat
caaaatttta gtattagttt ctgggtaagg 2820attcctaaac actagaaacc
tatgaatcat aatcgggaat agactataat aaattgtatg 2880gggaataata
attcgggatg gaaaatatca cttagaactg ttagagattg tgaaataatt
2940tggactttac aagatacttc tggaaataag gaaaatttaa tttttaggta
tgaagaactt 3000aataggatat ctaattatat aaataaatgg atttttgtaa
ctattactaa taatagatta 3060ggcaattcta gaatttagat caatggaaat
ttaatagttg aaaaatcaat ttcgaattta 3120ggtgatattc atgttagtga
taatatatta tttaaaattg ttggttgtga tgatgaaacg 3180tatgttggta
taagatattt taaagttttt aatacggaat tagataaaac agaaattgag
3240actttatata gtaatgagcc agatccaagt atcttaaaaa actattgggg
aaattatttg 3300ctatataata aaaaatatta tttattcaat ttactaagaa
aagataagta tattactctg 3360aattcaggca ttttaaatat taatcaacaa
agaggtgtta ctgaaggctc tgtttttttg 3420aactataaat tatatgaagg
agtagaagtc attataagaa aaaatggtcc tatagatata 3480tctaatacag
ataattttgt tagaaaaaac gatctagcat acattaatgt agtagatcgt
3540ggtgtagaat atcggttata tgctgataca aaatcagaga aagagaaaat
aataagaaca 3600tctaatctaa acgatagctt aggtcaaatt atagttatgg
attcaatagg aaataattgc 3660acaatgaatt ttcaaaacaa taatgggagc
aatataggat tactaggttt tcattcaaat 3720aatttggttg ctagtagttg
gtattataac aatatacgaa gaaatactag cagtaatgga 3780tgcttttgga
gttctatttc taaagagaat ggatggaaag aatga 382593894DNAArtificial
SequenceDescription 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 9atgccagtta atataaaaaa
ctttaattat aatgacccta ttaataatga tgacattatt 60atgatggaac cattcaatga
cccagggcca ggaacatatt ataaagcttt taggattata 120gatcgtattt
ggatagtacc agaaaggttt acttatggat ttcaacctga ccaatttaat
180gccagtacag gagtttttag taaagatgtc tacgaatatt aggatccaac
ttatttaaaa 240accgatgctg aaaaagataa atttttaaaa acaatgatta
aattatttaa tagaattaat 300tcaaaaccat caggacagag attactggat
atgatagtag atgctatacc ttatcttgga 360aatgcatcta caccgcccga
caaatttgca gcaaatgttg caaatgtatc tattaataaa 420aaaattatcc
aacctggagc tgaagatcaa ataaaaggtt taatgacaaa tttaataata
480tttggaccag gaccagttct aagtgataat tttactgata gtatgattat
gaatggccat 540tccccaatat cagaaggatt tggtgcaaga atgatgataa
gattttgtcc tagttgttta 600aatgtattta ataatgttca ggaaaataaa
gatacatcta tatttagtag acgcgcgtat 660tttgcagatc cagctctaac
gttaatgcat gaacttatac atgtgttaca tggattatat 720ggaattaaga
taagtaattt accaattact ccaaatacaa aagaattttt catgcaacat
780agcgatcctg tacaagcaga agaactatat acattcggag gacatgatcc
tagtgttata 840agtccttcta cggatatgaa tatttataat aaagcgttac
aaaattttca agatatagct 900aataggctta atattgtttc aagtgcccaa
gggagtggaa ttgatatttc cttatataaa 960caaatatata aaaataaata
tgattttgtt gaagatccta atggaaaata tagtgtagat 1020aaggataagt
ttgataaatt atataaggcc ttaatgtttg gctttactga aactaatcta
1080gctggtgaat atggaataaa aactaggtat tcttatttta gtgaatattt
gccaccgata 1140aaaactgaaa aattgttaga caatacaatt tatactcaaa
atgaaggctt taacatagct 1200agtaaaaatc tcaaaacgga atttaatggt
cagaataagg cggtaaataa agaggcttat 1260gaagaaatca gcctagaaca
tctcgttata tatagaatag caatgtgcaa gcctgtaatg 1320tacaaaaata
ccggtaaatc tgaacagtgt attattgtta ataatgagga tttatttttc
1380atagctaata aagatagttt ttcaaaagat ttagctaaag cagaaactat
agcatataat 1440acacaaaata atactataga aaataatttt tctatagatc
agttgatttt agataatgat 1500ttaagcagtg gcatagactt accaaatgaa
aacacagaac catttacaaa ttttgacgac 1560atagatatcc ctgtgtatat
taaacaatct gctttaaaaa aaatttttgt ggatggagat 1620agcctttttg
aatatttaca tgctcaaaca tttccttcta atatagaaaa tctacaacta
1680acgaattcat taaatgatgc tttaagaaat aataataaag tctatacttt
tttttctaca 1740aaccttgttg aaaaagctaa tacagttgta ggtgcttcac
tttttgtaaa ctgggtaaaa 1800ggagtaatag atgattttac atctgaatcc
acacaaaaaa gtactataga taaagtttca 1860gatgtatcca taattattcc
ctatatagga cctgctttga atgtaggaaa tgaaacagct 1920aaagaaaatt
ttaaaaatgc ttttgaaata ggtggagccg ctatcttaat ggagtttatt
1980ccagaactta ttgtacctat agttggattt tttacattag aatcatatgt
aggaaataaa 2040gggcatatta ttatgacgat atccaatgct ttaaagaaaa
gggatcaaaa atggacagat 2100atgtatggtt tgatagtatc gcagtggctc
tcaacggtta atactcaatt ttatacaata 2160aaagaaagaa tgtagaatgc
tttaaataat caatcacaag caatagaaaa aataatagaa 2220gatcaatata
atagatatag tgaagaagat aaaatgaata ttaacattga ttttaatgat
2280atagatttta aacttaatca aagtataaat ttagcaataa acaatataga
tgattttata 2340aaccaatgtt ctatatcata tctaatgaat agaatgattc
cattagctgt aaaaaagtta 2400aaagactttg atgataatct taagagagat
ttattggagt atatagatac aaatgaacta 2460tatttacttg atgaagtaaa
tattctaaaa tcaaaagtaa atagacacct aaaagacagt 2520ataccatttg
atctttcact atataccaag gacacaattt taatacaagt ttttaataat
2580tatattagta atattagtag taatgctatt ttaagtttaa gttatagagg
tgggcgttta 2640atagattcat ctggatatgg tgcaactatg aatgtaggtt
cagatgttat ctttaatgat 2700ataggaaatg gtcaatttaa attaaataat
tctgaaaata gtaatattac ggcacatcaa 2760agtaaattcg ttgtatatga
tagtatgttt gataatttta gcattaactt ttgggtaagg 2820actcctaaat
ataataataa tgatatacaa acttatcttc aaaatgagta tacaataatt
2880agttgtataa aaaatgactc aggatggaaa gtatctatta agggaaatag
aataatatgg 2940acattaatag atgttaatgc aaaatctaaa tcaatatttt
tcgaatatag tataaaagat 3000aatatatcag attatataaa taaatggttt
tccataacta ttactaatga tagattaggt 3060aacgcaaata tttatataaa
tggaagtttg aaaaaaagtg aaaaaatttt aaacttagat 3120agaattaatt
ctagtaatga tatagacttc aaattaatta attgtacaga tactactaaa
3180tttgtttgga ttaaggattt taatattttt ggtagagaat taaatgctac
agaagtatct 3240tcactatatt ggattcaatc atctacaaat actttaaaag
atttttgggg gaatccttta 3300agataggata cacaatacta tctgtttaat
caaggtatgc aaaatatcta tataaagtat 3360tttagtaaag cttctatggg
ggaaactgca ccacgtacaa actttaataa tgcagcaata 3420aattatcaaa
atttatatct tggtttacga tttattataa aaaaagcatc aaattctcgg
3480aatataaata atgataatat agtcagagaa ggagattata tatatcttaa
tattgataat 3540atttctgatg aatcttagag agtatatgtt ttggtgaatt
ctaaagaaat tcaaactcaa 3600ttatttttag cacccataaa tgatgatcct
acgttctatg atgtactaca aataaaaaaa 3660tattatgaaa aaacaacata
taattgtcag atactttgcg aaaaagatac taaaacattt 3720gggctgtttg
gaattggtaa atttgttaaa gattatggat atgtttggga tacctatgat
3780aattattttt gcataagtca gtggtatctc agaagaatat ctgaaaatat
aaataaatta 3840aggttgggat gtaattggca attcattccc gtggatgaag
gatggacaga ataa 38941023DNAArtificial SequenceDescription of
Artificial Sequence oligonucleotide used to introduce Stu I and
EcoR I into BoNT/A-L chain gene fragments 10aaaggccttt tgttaataaa
caa 231126DNAUnknown OrganismDescription of Artificial Sequence
oligonucleotide used to introduce Stu I and EcoR I into BoNT/A-L
chain gene fragment 11ggaattctta cttattgtat ccttta
261213PRTArtificial SequenceDescription of Artificial Sequence
polypeptide fragment used to raise antibodies 12Cys Ala Asn Gln Arg
Ala Thr Lys Met Leu Gly Ser Gly 1 5 10
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