U.S. patent application number 12/187189 was filed with the patent office on 2009-01-15 for methods for treating neurogenic inflammation.
This patent application is currently assigned to Allergan, Inc.. Invention is credited to Stephen Donovan.
Application Number | 20090017071 12/187189 |
Document ID | / |
Family ID | 27803690 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017071 |
Kind Code |
A1 |
Donovan; Stephen |
January 15, 2009 |
METHODS FOR TREATING NEUROGENIC INFLAMMATION
Abstract
The present invention relates to methods for treating neurogenic
inflammation pain. The methods include administering an effective
amount of a composition which includes a botulinum toxin component
and a substance P component to a patient, thereby treating the
neurogenic inflammation pain.
Inventors: |
Donovan; Stephen;
(Capistrano Beach, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
Allergan, Inc.
|
Family ID: |
27803690 |
Appl. No.: |
12/187189 |
Filed: |
August 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11284505 |
Nov 22, 2005 |
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12187189 |
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10082691 |
Feb 25, 2002 |
7022329 |
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11284505 |
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Current U.S.
Class: |
424/239.1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/046 20130101;
A61K 38/4893 20130101; A61K 38/046 20130101; A61K 38/4893 20130101;
A61P 29/00 20180101 |
Class at
Publication: |
424/239.1 |
International
Class: |
A61K 39/08 20060101
A61K039/08; A61P 29/00 20060101 A61P029/00 |
Claims
1. A method for treating neurogenic inflammation in a patient, the
method comprising the step of administering a therapeutically
effective amount of an agent to the patient, the administration
reducing the secretion of inflammation producing molecules thereby
treating the neurogenic inflammation, wherein the agent comprises a
botulinum toxin proteolytic domain, a botulinum toxin heavy chain
H.sub.N and a targeting moiety, wherein the botulinum toxin light
chain, the botulinum toxin heavy chain H.sub.N and the targeting
moiety are covalently linked together, and wherein the targeting
moiety is a substance P or a substance P component effective in
binding to a substance P receptor.
2. The method according to claim 1, wherein the botulinum toxin
proteolytic domain is a botulinum toxin serotype A proteolytic
domain, a botulinum toxin serotype B proteolytic domain, a
botulinum toxin serotype C1 proteolytic domain, a botulinum toxin
serotype D proteolytic domain, a botulinum toxin serotype E
proteolytic domain, a botulinum toxin serotype F proteolytic
domain, or a botulinum toxin serotype G proteolytic domain.
3. The method according to claim 1, wherein the botulinum toxin
heavy chain H.sub.N is a botulinum toxin serotype A heavy chain
H.sub.N, a botulinum toxin serotype B heavy chain H.sub.N, a
botulinum toxin serotype C, heavy chain H.sub.N, a botulinum toxin
serotype D heavy chain H.sub.N, a botulinum toxin serotype E heavy
chain H.sub.N, a botulinum toxin serotype F heavy chain H.sub.N, or
a botulinum toxin serotype G heavy chain H.sub.N.
4. The method according to claim 1, wherein the botulinum toxin
proteolytic domain comprises a botulinum toxin serotype A
proteolytic domain.
5. The method according to claim 1, wherein the botulinum toxin
heavy chain H.sub.N comprises a botulinum toxin serotype A heavy
chain H.sub.N.
6. The method according to claim 1, wherein the agent is
administered systemically.
7. The method according to claim 6, wherein the systemic
administration is oral administration, intravenous administration,
or administration as an inhalant.
8. The method according to claim 1, wherein the agent is
administered subcutaneously or intramuscularly.
9. A method for treating neurogenic inflammation in a patient, the
method comprising the step of administering a therapeutically
effective amount of an agent to the patient, the administration
reducing the secretion of inflammation producing molecules thereby
treating the neurogenic inflammation, wherein the agent comprises
botulinum neurotoxin LH.sub.N fragment covalently linked to a
targeting moiety, and wherein the targeting moiety is a substance P
or a substance P component effective in binding to a substance P
receptor.
10. The method according to claim 9, wherein the botulinum
neurotoxin LH.sub.N component is a botulinum neurotoxin serotype A
LH.sub.N fragment, a botulinum neurotoxin serotype B LH.sub.N
fragment, a botulinum neurotoxin serotype C.sub.1 LH.sub.N
fragment, a botulinum neurotoxin serotype D LH.sub.N fragment, a
botulinum neurotoxin serotype E LH.sub.N fragment, a botulinum
neurotoxin serotype F LH.sub.N fragment, or a botulinum neurotoxin
serotype G LH.sub.N fragment.
11. The method according to claim 9, wherein the botulinum toxin
heavy chain LH.sub.N fragment comprises a botulinum toxin serotype
A heavy chain LH.sub.N fragment.
12. The method according to claim 9, wherein the agent includes one
or more spacer regions between the LH.sub.N fragment and the
targeting moiety.
13. The method according to claim 9, wherein the agent is
administered systemically.
14. The method according to claim 13, wherein the systemic
administration is oral administration, intravenous administration,
or administration as an inhalant.
15. The method according to claim 9, wherein the agent is
administered subcutaneously or intramuscularly.
Description
[0001] This application is a continuation and claims priority
pursuant to 35 U.S.C. .sctn. 120 to U.S. patent application Ser.
No. 11/284,505, filed Nov. 22, 2005, a divisional that claims
priority pursuant to 35 U.S.C. .sctn. 120 to U.S. patent
application Ser. No. 10/082,691, filed Feb. 25, 2002, now U.S. Pat.
No. 7,022,329, each of which is hereby incorporated by reference in
its entirety.
BACKGROUND
[0002] The present invention relates to methods for treating
inflammation pain, for example, neurogenic inflammation pain. In
particular, the present invention relates to methods for treating
pain using Clostridial toxin compositions and methods for making
the Clostridial toxin compositions.
[0003] In recent years attention has been directed toward the
interaction between components of the nervous system and target
cells of the immune system. Communication between nerve cells and
cells which mediate inflammation is demonstrative of such
neuroimmune interactions. For example, several studies have
demonstrated that mast cells and vascular endothelial cells are
often in close contact with nerves and that there may be a
functional interaction between these cells and the nervous system.
In addition, recent evidence suggests that the neuropeptide
substance P is an important mediator in cross talk between nerves
and inflammation mediating cells.
[0004] Mast cells and vascular endothelial cells present a rich
source of inflammation mediating compounds which include histamine,
prostaglandins, leukotrienes, neutral proteases, cytokines,
bradykinin and nitric oxide. It is postulated that in response to
stimulus, substance P may be released from nerve cells and then
bind to inflammation mediating cells subsequently triggering
release of these and/or other compounds resulting in inflammation.
This inflammation is thought to produce pain which includes
fibromyalgia pain, migraine headache, arthritis pain, interstitial
cystitis pain, myofascial pain syndrome and irritable bowel
pain.
[0005] What is needed are methods to stop or reduce the occurrence
of inflammation and inflammation related pain which occurs in
response to neural stimulus.
Botulinum Toxin
[0006] The anaerobic, gram positive bacterium Clostridium botulinum
produces a potent polypeptide neurotoxin, botulinum toxin, which
causes a neuroparalytic illness in humans and animals referred to
as botulism. The spores of Clostridium botulinum are found in soil
and can grow in improperly sterilized and sealed food containers of
home based canneries, which are the cause of many of the cases of
botulism. The effects of botulism typically appear 18 to 36 hours
after eating the foodstuffs infected with a Clostridium botulinum
culture or spores. The botulinum toxin can apparently pass
unattenuated through the lining of the gut and attack peripheral
motor neurons. Symptoms of botulinum toxin intoxication can
progress from difficulty walking, swallowing, and speaking to
paralysis of the respiratory muscles and death.
[0007] Botulinum toxin type A ("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 LD.sub.50
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.sub.1, D, E, F and G each of
which is distinguished by neutralization with serotype-specific
antibodies. The different serotypes of botulinum toxin vary in the
animal species that they affect and in the severity and duration of
the paralysis they evoke. For example, it has been determined that
BoNt/A is 500 times more potent, as measured by the rate of
paralysis produced in the rat, than is botulinum toxin serotype B
(BoNT/B). Additionally, botulinum toxin type B ("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.
[0008] Botulinum toxins have been used in clinical settings for the
treatment of neuromuscular disorders characterized by hyperactive
skeletal muscles. Botulinum toxins, and modified botulinum toxins,
are also useful for the treatment of pain, see for example, U.S.
Pat. Nos. 6,113,915; 6,333,037; 6,235,289; 5,714,468 and; WO
94/15629 each of which is incorporated in its entirety herein by
reference.
[0009] BoNt/A has been approved by the U.S. Food and Drug
Administration for the treatment of blepharospasm, strabismus,
hemifacial spasm and cervical dystonia. Additionally a botulinum
toxin type B has been approved by the FDA for the treatment of
cervical dystonia. 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.
[0010] 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.
[0011] Regardless of serotype, the molecular mechanism of toxin
intoxication appears to be similar and to involve at least three
steps or stages. In the first step of the process, the toxin binds
to the presynaptic membrane of the target neuron through a specific
interaction between the H chain and a cell surface receptor; the
receptor is thought to be different for each serotype of botulinum
toxin and for tetanus toxin. The carboxyl end segment of the H
chain, H.sub.c, appears to be important for targeting of the toxin
to the cell surface.
[0012] 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.
[0013] 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.
[0014] 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.sub.1 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 hemagglutinin protein and a non-toxin and
non-toxic nonhemagglutinin 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.
[0015] 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.
[0016] 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.
[0017] It has been reported (as exemplary examples) that BoNt/A has
been used clinically as follows:
[0018] (1) about 75-125 units of BOTOX.RTM..sup.1 per intramuscular
injection (multiple muscles) to treat cervical dystonia;
.sup.1Available from Allergan, Inc., of Irvine, Calif. under the
tradename BOTOX.RTM..
[0019] (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);
[0020] (3) about 30-80 units of BOTOX.RTM. to treat constipation by
intrasphincter injection of the puborectalis muscle;
[0021] (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.
[0022] (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).
[0023] (6) to treat upper limb spasticity following stroke by
intramuscular injections of BOTOX.RTM. into five different upper
limb flexor muscles, as follows: [0024] (a) flexor digitorum
profundus: 7.5 U to 30 U [0025] (b) flexor digitorum sublimus: 7.5
U to 30 U [0026] (c) flexor carpi ulnaris: 10 U to 40 U [0027] (d)
flexor carpi radialis: 15 U to 60 U [0028] (e) biceps brachii: 50 U
to 200 U. Each of the five indicated muscles has been injected at
the same treatment session, so that the patient receives from 90 U
to 360 U of upper limb flexor muscle BOTOX.RTM. by intramuscular
injection at each treatment session.
[0029] 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.
SUMMARY
[0030] The present invention provides for methods to treat various
types of inflammation and pain associated with inflammation.
[0031] In accordance with the present invention there are provided
methods for treating neurogenic inflammation and neurogenic
inflammation pain. The methods may include administering an
effective amount of a composition which includes a botulinum toxin
component and a substance P component to a patient thereby treating
the neurogenic inflammation and/or neurogenic inflammation
pain.
[0032] The botulinum toxin component may include an H.sub.N and an
L chain. For example, the H.sub.N may be obtained from a botulinum
toxin serotype A, serotype B, serotype C, serotype D, serotype E,
serotype F or serotype G. Also for example, the L chain may be
obtained from a botulinum toxin serotype A, serotype B, serotype C,
serotype D, serotype E, serotype F or serotype G.
[0033] In accordance with the invention the substance P component
may be a substance P, a precursor of substance P or a substance P
analogue.
[0034] The present invention provides for methods for treating pain
caused by inflammation including fibromyalgia pain, myofascial pain
syndrome pain, arthritis pain, migraine headache pain, irritable
bowel syndrome pain, Crohn's disease pain and interstitial cystitis
pain.
[0035] In accordance with the present invention, the compositions
may be administered subcutaneously, intramuscularly or
systemically. The compositions may be administered with a needle,
by needleless injection orally, by inhalable delivery methods such
as an inhalable mist or like methods.
[0036] Methods of the present invention provide for pain relief
ranging from about 5% to about 100% in effectiveness. For example,
pain may be reduced by about 20%, about 40%, about 50%, about 60%,
about 80% or about 100%.
[0037] The present invention also provides for methods for
inhibiting pain caused by degranulation of mast cells and/or
release of inflammation mediating compounds from vascular
endothelial cells. The method may include administering to a
patient an effective amount of a composition which includes a
botulinum toxin component attached to a substance P component,
thereby inhibiting pain caused by degranulation of mast cells
and/or release of inflammation mediating compounds from vascular
endothelial cells. For example, the present invention provides for
methods for inhibiting pain caused by histamine release from mast
cells. These methods may include administering to a patient an
effective amount of a composition which includes a botulinum toxin
component attached to a substance P component, thereby inhibiting
pain caused by histamine release from mast cells.
[0038] Each and every feature described herein, and each and every
combination of two or more of such features, is included within the
scope of the present invention provided that the features included
in such a combination are not mutually inconsistent.
DEFINITIONS
[0039] An "agent" is defined as a modified neurotoxin that
possesses some or all of the biological activity biological
activity of an unmodified neurotoxin. Modified neurotoxins include
variants and fragments of neurotoxins. One example of a modified
neurotoxin, as disclosed herein, is a portion of a botulinum toxin
coupled to a substance P molecule.
[0040] A "clostridial neurotoxin" may refer to an intact toxin for
example a botulinum toxin, butyricum toxin or tetani toxin or a
fragment or portion of a toxin for example a fragment or portion of
a botulimum toxin, butyricum toxin or tetani toxin.
[0041] "H.sub.C" means a fragment obtained from the H chain of a
Clostridial toxin which is equivalent, for example functionally
equivalent, to the carboxyl end fragment of the H chain, or the
portion corresponding to that fragment in the intact H chain
involved in binding to a cell surface or cell surface receptor.
[0042] "H.sub.N" means a fragment or variant obtained from an H
chain of a Clostridial toxin which may be functionally equivalent
to the portion of an intact H chain involved in the translocation
of at least the L chain across an intracellular endosomal membrane
into a cytoplasm of a cell. An H.sub.N, may result from an H.sub.c
being removed from an H chain. An H.sub.N may also result from an H
chain being modified such that its H.sub.C no longer binds to
cholinergic cell surfaces.
[0043] "Heavy chain" means the heavy chain of a clostridial
neurotoxin or a fragment or variant of an H.sub.N of a clostridial
neurotoxin. A heavy chain may have a molecular weight of about 100
kDa and can be referred to as H chain, or as H.
[0044] "LH.sub.N" means a fragment obtained from a clostridial
neurotoxin that contains the L chain coupled to an H.sub.N.
LH.sub.N can be obtained from the intact clostridial neurotoxin by
proteolysis, so as to remove or to modify the H.sub.C domain.
[0045] "Light chain" means the light chain of a clostridial
neurotoxin or a fragment or variant of a light chain of a
clostridial neurotoxin. A light chain may have 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.
[0046] "Linker" means a molecule which couples two or more other
molecules or components together.
[0047] "Local administration" means direct administration by a
non-systemic route at or in the vicinity of the site of an
affliction, disorder, or perceived pain.
[0048] "Neurogenic" means arising from the nervous system. For
example, neurogenic inflammation refers to inflammation that may be
caused, at least in part, directly or indirectly, by the nervous
system.
[0049] "Spacer" means a molecule or set of molecules which
physically separate and add distance between the components. One
function of a spacer may be to prevent steric hindrance between the
components.
[0050] "Substantial or Substantially" means largely but not
entirely. For example, substantial relief of pain may mean pain
relief of 10%, 20%, 30%, 40% 50% or more.
[0051] "Targeting moiety" means a molecule that has a specific
binding affinity for a cell surface or cell surface receptor.
[0052] "Variable region" means the part of an antibody that varies
extensively from one antibody to another as a result of alternative
subunit sequences.
[0053] "Variant" means a molecule or peptide which is substantially
the same as that of a disclosed molecule or peptide in its
structure and function. For example, a variant of a specified light
chain may have non-consequential amino acid sequence substitutions
when compared to the amino acid sequence of the specified light
chain. Variants may be considered to be equivalent to the
specifically disclosed molecules and as such are within the scope
of the invention.
DESCRIPTION
[0054] This invention is based upon the discovery that inflammation
pain can be treated by administering to a patient an agent which
includes a clostridial neurotoxin component and a targeting moiety
component, wherein the targeting moiety component may bind to cells
involved in mediating inflammation.
[0055] The mechanism of action for these agents in alleviating pain
is currently not fully understood. However, without wishing to
limit the invention to any particular theory or mechanism of
operation, it is believed that the agents disclosed herein may
target cells, for example, mast cells and/or vascular endothelial
cells which may have receptors for the targeting moieties, for
example, a substance P targeting moiety.
[0056] Mast cells and vascular endothelial cells present many
distinct biologically active, inflammation mediators that may
include histamine, prostaglandins, leukotrienes, neutral proteases,
cytokines bradykinin and nitric oxide. Release of these and/or
other mediators may contribute to the events which cause neurogenic
inflammation. Further examples of mediators that may contribute to
neurogenic inflammation are discussed in First (U.S. Pat. No.
6,063,768) which is incorporated in its entirety herein by
reference.
[0057] Sensory neurons containing substance P are thought to be
involved in regulating the inflammatory and immune response in the
peripheral tissues that they innervate. It is theorized that these
sensory neurons release substance P which binds to and causes
secretion from inflammation mediating cells. For example, substance
P may bind to mast cell receptor sites thereafter triggering
degranulation which includes secretion from mast cells of
histamine, prostiglandins, leukotrines, serotonin and other
molecules that may serve as inflammatory mediators (Spanos et al. J
of Urology Vol 157 p 669-672; Toyoda et al Arch Dermatol Res Vol
292 p 418-421). In another example, substance P may trigger release
of inflammation mediators such as bradykinin, nitric oxide and
vasoactive intestinal peptide from vascular endothelial cells.
[0058] It is theorized that inflammatory mediator release triggered
by substance P begins with the initial binding of substance P to
specific receptors which may be mobile and randomly distributed on
the membrane surface of certain cells, for example, mast cells
and/or vascular endothelial cells. These substance P-receptor
complexes may accumulate in specialized regions of the membrane
termed coated pits. From this stage, receptor mediated endocytosis
(RME) may proceed to the formation of smooth-walled vesicles which
allow entry of the substance P-receptor complexes into the cell.
These vesicles, often referred to as "endosomes" or "receptosomes,"
may fuse together or combine to produce larger vesicles.
Subsequently, the internal pH of the endosomes may be decreased by
the action of proton pumps. This decrease in pH may change the
conformation of the receptor and/or substance P resulting in the
release of the substance P from the receptor and the formation of
separate receptor-containing vesicles and substance P-containing
containing vesicles. The resulting substance P vesicles along with
the internalized substance P, may be delivered to and fused with
lysosomes where the eventual breakdown of the vesicles and release
of the substance P likely takes place.
[0059] It is thought that at some point after formation of the
substance P-receptor complex, possibly after internalization of the
complex, release of inflammation mediators is triggered.
[0060] During secretion or exocytosis, the mediators may be
included in vesicles which fuse to the inner surface of the cell
membrane thereby releasing the vesicle contents to the outside of
the cell. Without wishing to limit the present invention to any
theory or mechanism of operation, it is theorized that interference
with the exocytosis process may be the mode of action of the agents
of the present invention.
[0061] It is theorized that agents of the present invention may
operate by first targeting inflammation mediating cells by use of a
targeting moiety component such as substance P, and then prevent or
reduce the secretion of inflammation producing molecules by
cleaving or by otherwise interfering with the function of proteins
involved in the secretory process by use of a light chain
component, for example, a botulinum light chain component. A heavy
chain component, for example H.sub.N, may also function in certain
embodiments of the present invention by, for example, assisting in
the release of an agent of the invention from intracellular
vesicles, for example, endosomes.
[0062] The agents described for use in this invention may be very
specific for treating inflammation pain without being cytotoxic
because they may not substantially or significantly interact with
and/or interfere with neurons. Also, the agents can alleviate pain
without being cytotoxic to their target neurons. Because of lack of
neural interaction and lack of cytotoxicity, agents within the
scope of the present invention can be administered either locally
at or near sites of inflammation or inflammation pain or
systemically.
[0063] Agents may be used to treat conditions cause by
inflammation, for example neurogenic inflammation. Examples of
conditions caused by neurogenic inflammation that may be treated by
methods of the present invention include pain associated with
fibromyalgia, certain forms of arthritis, myofascial pain syndrome,
interstitial cystitis and irritable bowel syndrome. Additional
examples of conditions that may be treated in accordance with the
present invention include myasthenia gravis, systemic lupus
erythematosus, discoid lupus erythematosus, organ transplant,
tissue transplant, fluid transplant, Graves disease,
thyrotoxicosis, autoimmune diabetes, haemolytic anaemia,
thrombocytopenic purpura, neutropenia, chronic autoimmune
hepatitis, autoimmune gastritis, pernicious anaemia, Hashimoto's
thyroiditis, Addison's disease, Sjogren's syndrome, primary biliar
cirrhosis, polymyositis, scleroderma, systemic sclerosis, pemphigus
vulgaris, bullous pemphigoid, myocarditis, rheumatic carditis,
glomerulonephritis, uveitis, orchitis, ulcerative colitis,
vasculitis, atrophic gastritis, pernicious anaemia, type 1 diabetes
mellitus. Further examples of conditions that may be caused by
neurogenic inflammation that may be treated by methods of the
present invention are discussed in First (U.S. Pat. No.
6,063,768).
[0064] According to one broad aspect of the invention, methods are
provided for the use of a clostridial neurotoxin component
covalently coupled to a targeting moiety. The clostridial
neurotoxin component may be obtained from Clostridium beratti,
Clostridium butyricum, or Clostridium botulinum, for example,
Clostridium botulinum toxin types A, B, C, D, E, F and G may be
used.
[0065] The clostridial neurotoxin component may include only a
fragment of the entire neurotoxin. For example, it is known in the
art that the H.sub.c of the neurotoxin molecule can be removed from
the other segment of the H chain, the H.sub.N, such that the
H.sub.N fragment remains disulphide linked to the L chain of the
neurotoxin molecule to provide a fragment known as the LH.sub.N. In
addition, LH.sub.N can be produced by modifying the H.sub.c
fragment to reduce or eliminate the targeting affinity or ability
of H.sub.C to bind to its native target site. H.sub.C can be
modified by methods known in the art, for example, alterations to
the amino acid sequence of the H.sub.c or chemical modification to
the H.sub.c. Furthermore, the light chain or a fragment of the
light chain, with no associated H chain or H chain fragment, can be
coupled to a targeting moiety in accordance with the present
invention. Therefore, in one embodiment of the present invention
LH.sub.N of a clostridial neurotoxin is covalently coupled to a
targeting moiety, for example, substance P. In another embodiment,
the L chain of a clostridial neurotoxin is covalently coupled to a
targeting moiety, for example, substance P.
[0066] In another embodiment, the agent includes an H chain of a
clostridial neurotoxin, in which the H.sub.c is removed, mutated or
chemically modified to reduce or eliminate the ability of H.sub.c
to bind to the neurotoxin receptors at the neuromuscular junction
combined with the L-chain of a different clostridial neurotoxin, to
form a hybrid. For example, in one embodiment, the clostridial
neurotoxin component comprises an H chain with the H.sub.c removed,
mutated or chemically modified obtained from botulinum toxin type
A, and an L chain obtained from another botulinum toxin type. The
described hybrid is covalently coupled to a targeting moiety.
[0067] In another embodiment, the agent includes an L chain of a
clostridial neurotoxin, or a fragment of the L chain containing the
endopeptidase activity, linked to a targeting moiety.
[0068] In a broad embodiment, spacers may be used to physically
separate components of agents used in accordance with the present
invention. For example, an agent of the present invention may
comprise an L chain or a LH.sub.N connected to a targeting moiety,
for example substance P, through a spacer. A spacer may function to
create a distance between the components to minimize or eliminate
steric hindrance of the components that may otherwise occur.
[0069] In one embodiment, the spacer region is made up of sugar
molecules, for example, saccharides, glucose, etc. In another
embodiment, the spacer region may be constructed from an aliphatic
chain. In another embodiment, the spacer regions may be constructed
by linking together a series of amino acids, preferably glycine
because glycine are small and are devoid of any functional group.
In yet another embodiment, the spacer region may comprise one or
more of the sugar molecules, aliphatic chains, and amino acids.
[0070] In one embodiment, a spacer comprises a proline, serine,
threonine and/or cysteine-rich amino acid sequence similar or
identical to an immunoglobulin hinge region. For example, the
spacer may comprise the amino acid sequence of a human
immunoglobulin g1 hinge region.
[0071] Spacers may comprise hydrocarbon moieties, for example,
hydrocarbon moieties represented by the chemical formulas:
HOOC--(CH.sub.2).sub.n--COOH, where n=1-12 or,
HO--(CH.sub.2).sub.n--COOH, where n>10
[0072] In one embodiment, linkers may be used to link together two
or more molecules, components and/or spacers. For example, a Linker
may be used to link a targeting moiety to a L or LH.sub.N. In
another embodiment, a Linker (Y) may be employed to link an L or
LH.sub.N to a spacer; in turn, that spacer may then be linked to
targeting moiety by another Linker (Y), forming, for example, an
agent comprising the structure:
L-Y-Spacer-Y-Targeting Moiety.
[0073] Examples of substances that can be used as linker (Y) are
2-iminothiolane, N-succinimidyl-3-(2-pyridyldithio) propionate
(SPDP), 4-succinimidyloxycarbonyl-alpha-(2-pyridyldithio)toluene
(SMPT), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl
4-(p-maleimidophenyl)butyrate (SMPB),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
bis-diazobenzidine and glutaraldehyde.
[0074] In one embodiment, a linker may be attached to an amino
group, a carboxylic group, a sulfhydryl group or a hydroxyl group
or an amino group of a component. For example, a linker may be
linked to a carboxyl acid group of an amino acid of a targeting
moiety.
[0075] Although the described chemistry may be used to couple
components of agents used in the invention, any other coupling
chemistry known to those skilled in the art capable of chemically
attaching one component to another component of an agent of the
invention is included within the scope of the present
invention.
[0076] In one embodiment, an agent comprises an H.sub.N and/or an L
chain and a targeting moiety, covalently linked together. For
example, the H.sub.N may be linked to the L chain and the L chain
may be linked to the targeting moiety. In another example, the
H.sub.N may be linked to the targeting moiety and to the L chain.
In still another example, the targeting moiety may be linked to
H.sub.N and the L chain. In still another example, the L chain may
be linked to the H.sub.N and the targeting moiety. Linkers and/or
spacers may be used between some, all or none of the components in
these examples.
[0077] In another embodiment, the targeting moieties may be
components that are substantially similar to substance P. These
components include substance P precursors, fragments and analogs.
The history, isolation, identification, and synthesis of substance
P and its precursors, fragments and analogs are disclosed in U.S.
Pat. No. 5,891,842 (incorporated herein by reference in its
entirety).
[0078] Substance P is an 11 amino acid peptide which has a number
of different natural and synthetic precursor forms; has been
demonstrated to be converted into a variety of naturally occurring
amino-terminal peptide fragments; and can be obtained in analog
format compromising, substituted counterparts thereof, for example,
lysine methyl ester, D-amino acids or disulfide bridges
substitutions, which may thereby yield more stable and
discriminating formulations. A representative listing of substance
P and its related chemical entities is provided by Table I below.
The amino acid sequence (1) in Table I
(Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide) can be referred
to as SEQ ID NO: 1, and the subsequent 17 amino acid sequences set
forth in Table one can be similarly identified as SEQ ID NO:2 to
SEQ ID NO:18.
TABLE-US-00001 TABLE 1 Substance P, and Representative Precursors,
Fragments and Stabilized Or Substituted Analogs SEQ ID Name Formula
NO: (1) Substance P
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-amide 1 Natural
Precursors: (2) Substance P-Glycine*
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly 2 (3) Substance
P-Glycine- Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 3
Lysine* Lys (4) Substance P-Glycine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 4 Lysine-Arginine*
Lys-Arg Carboxy-Ester Synthetic Precursors: (5) Substance P-Glycine
Methyl Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 5
Ester.degree. OMe (6) Substance P-Glycine-Lysine
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 6 Methyl
Ester.degree. Lys-OMe (7) Substance P-Glycine-Lysine
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 7 Arginine Methyl
Ester.degree. Lys-Arg-OMe (8) Substance P-Glycine Ethyl
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 8 Ester.degree.
OEth (9) Substance P-Glycine-Lysine
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 9 Ethyl
Ester.degree. Lys-OEth (10) Substance P-Glycine-
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly- 10 Lysine Arginine
Ethyl Ester.degree. Lys-Arg-OEth Naturally-Occurring Amino-Terminal
Peptide Fragments: (11) Substance P/1-4# Arg-Pro-Lys-Pro 11 (12)
Substance P/1-7# Arg-Pro-Lys-Pro-Gln-Gln-Phe 12 (13) Substance
P/1-9# Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly 13 Analogs Comprising
Synthetic D-Amino Acids Or Disulfide (Cys-Cys) Bridges: (14)
[D-Pro2, D-Phe7, D-Trp9]-
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-PheD-Trp-Leu-Met- 14 Substance
P.sup.c amide (15) [D-Pro2, D-Phe7, D-Trp9]-
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-Phe-D-Trp-Leu- 15 (Substance
P-Glycine.sup.c) Met-Gly (16) [D-Pro2, D-Trp7, D-Trp9]-
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met- 16 Substance
P.sup.c amide (17) [D-Pro2, D-Trp7, D-Trp9]-
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met- 17 Substance
P-Glycine.sup.c Gly (18) [Cys3, Cys6, Tyr8, Pro 10]-
Arg-Pro-Cys-Pro-Gln-Cys-Phe-Tyr-Gly-Pro-Met-amide 18 Substance
P.sup.c *Shimon et al., J. Neurochem. 59: 81-92 (1992) .degree.Lee
et al., Eur. J. Biochem. 114: 315-327 (1981); Pernow, B.,
Pharmacol. Rev. 35: 86-138 (1983); and Regoli et al., TIPS 9:
290-295 (1988). #Stewart et al., Nature 262: 784-785 (1986); and
Skilling et al., J. Neurosci. 10: 1309-1318 (1990) .sup.cLavielle
et al., Biochem. Pharmacol. 37: 41 (1988); and Quirion, R. and T.
V. Dam, Regulatory Peptides 22: 18 (1988)
[0079] In one embodiment, agents for use in accordance with the
invention comprise a hybrid of two clostridial neurotoxins. For
example, the H chain, in one embodiment H.sub.N, may be obtained
from botulinum toxin A, B, C, D, E, F or G and the L chain obtained
from another botulinum toxin selected from botulinum toxin A, B, C,
D, E, F or G. The two chains from different botulinum toxin
serotypes may be joined together by covalent bonds and/or by
disulfide bonds and/or by hydrogen bonds and/or by ionic bonds to
for a hybrid LH.sub.N. This hybrid LH.sub.N may be linked to a
targeting moiety, for example, substance P or a component that is
substantially similar to substance P, for example, substance P
precursors, substance P analogs and substance P fragments, by
standard methodologies known in the art. In one embodiment the
targeting moiety is covalently linked to a hybrid LH.sub.N.
[0080] In another embodiment, the agents comprises an L chain of a
clostridial neurotoxin, or a fragment of an L chain containing the
endopeptidase activity, coupled to substance P or component that is
substantially similar to substance P. The L chain or fragment of
the L chain is obtained from botulinum toxin A, B, C, D, E or G and
is coupled to substance P or components that are substantially
similar to substance P, for example, substance P precursors,
substance P analogs and substance P fragments, by standard
methodologies known in the art. Linkers and/or spacer components
may be used in the coupling.
[0081] An agent used in accordance with the present invention may
comprise a heavy chain or a portion of a heavy chain of a
clostridial neurotoxin. Even more preferably, the H.sub.N of the
heavy chain is able to facilitate the transfer of the agent across
an endosome membrane into the cytosol of a cell, for example, a
mast cell. The clostridial neurotoxin heavy chain may be obtained
from Clostridium botulinum neurotoxin type A. In other embodiments,
the heavy chain may be obtained from Clostridium botulinum types B,
C, D, E, F, G and mixtures thereof. Also, the heavy chain may be
obtained from neurotoxins obtained from Clostridium baratii and
Clostridium butyricum or Clostridium tetani.
[0082] Agents that may be used in accordance with the present
invention are also disclosed in U.S. patent application Ser. Nos.
09/489,667, 09/922,093 and 09/625,098 which are incorporated in
their entirety herein by reference.
[0083] According to another broad aspect of this invention,
recombinant DNA methodologies may be used to produce the components
of agents useful in accordance with the invention, including the
targeting moiety, the light chain or light chain fragment and/or
the heavy chain or heavy chain fragment. These techniques may
include steps of obtaining cloned genes from natural sources, or
from synthetic oligonucleotide sequences, which may encode
clostridial neurotoxin components including clostridial neurotoxin
heavy chains, light chains or variants thereof, modified
clostridial neurotoxin chains and/or fragments of the chains.
Cloned genes may also encode a targeting moiety.
[0084] The genes may be cloned into, for example, cloning vectors,
such as phages or plasmids or phagemids. The recombinant vectors
are transformed into host cells, for example, into a prokaryotic
cell, for example, E. coli. Proteins can be expressed and then
isolated using conventional techniques.
[0085] Fusion genes may be used which encode more than one
component of an agent. For example, a targeting moiety and a
botulinum toxin heavy chain and/or light chain and/or a fragment of
a heavy and/or a fragment of a light chain, can be produced from a
single cloned gene as a fusion protein. Alternatively, individual
components obtained from recombinant techniques can be chemically
coupled to other components obtain from other sources. For example,
a synthetic or naturally occurring targeting moiety component may
be coupled to a recombinant L chain or to a recombinant fusion
LH.sub.N. The linkages between the clostridial components and the
targeting moieties may include appropriate spacer components which
may also be DNA encoded and included in the fusion gene
construct.
[0086] In another embodiment, the required LH.sub.N, which may be a
hybrid of an L chain and an H.sub.N from different clostridial
toxin types, is expressed recombinantly as a fusion protein. Such
LH.sub.N hybrid may also be coupled to the targeting moiety, which
may further include one or more spacer regions between them.
[0087] In another embodiment of the invention the L chain of a
clostridial neurotoxin, or a fragment of the L chain containing the
endopeptidase activity, is expressed recombinantly as a fusion
protein with the H.sub.N of the H chain and the targeting moiety.
The expressed fusion protein may also include one or more spacer
regions. For example, the L chain may be fused to H.sub.N which is
in turn fused to the targeting moiety. In another example, the
H.sub.N may be fused to the L chain which is in turn fused to the
targeting moiety. Spacer components may be expressed recombinantly
between some or all of the components of an agent of the
invention.
[0088] In one embodiment of producing a hybrid of LH.sub.N, the L
chain is obtained from botulinum toxin type B and the amine end
segment of the H.sub.N chain fragment is obtained from botulinum
toxin type A. The H.sub.N fragment of the botulinum toxin type A is
produced according to the method described by Shone C. C.,
Hambleton, P., and Melling, J. (1987, Eur. J. Biochem. 167,
175-180) and the L chain of botulinum toxin type B according to the
method of Sathyamoorthy, V. and DasGupta, B. R. (1985, J. Biol.
Chem. 260, 10461-10466). The free cysteine on the amine end segment
of the H chain fragment of botulinum toxin type A is then
derivatized by the addition of a ten-fold molar excess of dipyridyl
disulphide followed by incubation at 4 degrees C. overnight. The
excess dipyridyl disulphide and the thiopyridone by product are
then removed by desalting the protein over a PD10 column
(Pharmacia) into PBS.
[0089] The derivatized H.sub.N is then concentrated to a protein
concentration in excess of 1 mg/ml before being mixed with an
equimolar portion of L chain from botulinum toxin type B (>1
mg/ml in PBS). After overnight incubation at room temperature the
mixture is separated by size exclusion chromatography over Superose
6 (Pharmacia), and the fractions analyzed by SDS-PAGE. The chimeric
LH.sub.N is then available to produce a conjugated agent which
includes a targeting moiety component.
[0090] The example described above is purely illustrative of the
invention. In synthesizing the agents, the coupling of the
targeting moieties to the clostridial components, for example the
modified clostridial neurotoxins or fragments thereof, may be
achieved via chemical coupling using reagents and techniques known
to those skilled in the art. Thus, any coupling chemistry capable
of covalently attaching the targeting moieties of the agents to
clostridial neurotoxin components and known to those skilled in the
art is covered by the scope of this application.
[0091] In one embodiment of the invention, there are provided
methods for the treatment of pain which comprise locally
administering directly to a region of inflammation and/or
inflammation pain in a patient, in therapeutically effective doses,
an agent of the invention. In another embodiment, the invention
provides for administration of an agent of the invention near the
site of inflammation and/or inflammation pain in a patient, for
example, within about 0.1 to about 100 cm, or for example, about 1
cm to about 10 cm from the site of inflammation and/or inflammation
pain.
[0092] Known local drug administration methods suitable for
administration may be used including injection, with or without the
use of a needle, and by insertion of a controlled release implant.
Routes of administration include, without limitation, transdermal,
peritoneal, subcutaneous, intramuscular, and intrarectal
injection.
[0093] In another embodiment, a therapeutically effective dose of
an agent of the invention is administered by systemic
administration, for example, oral administration, intravenous
administration or administration as an inhalant.
[0094] An agent, such as botulinum toxin component-substance P or
components that are substantially similar to substance P can
require, according to the methods of the present invention, from
about 1 to 7 days to begin to achieve an effect upon a site of
inflammation pain.
[0095] The amount of the agents administered can vary widely
according to the particular disorder being treated, its severity
and other various patient variables including size, weight, age,
and responsiveness to therapy.
[0096] Methods of the present invention provide for pain relief
ranging from about 5% to about 100% in effectiveness. For example,
pain may be reduced by about 20%, about 40%, about 50%, about 60%,
about 80% or about 100%.
[0097] Methods for determining the appropriate route of
administration and dosage are generally determined on a
case-by-case basis by the attending physician. Such determinations
are routine to one of ordinary skill in the art (see for example,
Harrison's Principles of Internal Medicine (1998), edited by
Anthony Fauci et al., 14.sup.th edition, published by McGraw
Hill).
[0098] Generally, the dose of an agent to be administered will vary
with the age, presenting condition and weight of the patient to be
treated. The potency of the agent will also be considered. Agent
(e.g. conjugate) potency is expressed as a multiple of the
LD.sub.50 value of an agent of the invention for a mouse. A "U" or
"unit" of an agent can be defined as the amount of toxin that kills
50% of a group of mice that were disease-free prior to inoculation
with the agent. Alternatively, potency may be expressed as the
LD.sub.50 value of an agent that would be produced by an equal
molar amount of botulinum toxin with a functional H.sub.C.
[0099] Agents of the invention can be administered in a dose of
about 0.01 units up to about 1,000 units. In one embodiment,
individual dosages of about 1 unit to about 30 units are used. In
another embodiment, individual dosages of about 30 units to about
60 units are used. In still another embodiment, individual dosages
of about 60 units to about 180 units are used. Generally, the
agents may be administered as a composition at a dosage that is
proportionally equivalent to, for example, about 2.5 cc/100 units.
Those of ordinary skill in the art will know, or can readily
ascertain, how to adjust these dosages for an agent of greater or
lesser potency.
[0100] Preferably, the lowest therapeutically effective dosage will
be administered to the patient. The lowest therapeutic dosage is
that dosage which results in detection by the patient of a
reduction in the occurrence and/or magnitude of pain experienced by
the patient, for example, pain experience by a patient which is
associated with neurogenic inflammation.
[0101] Methods for assessing or quantifying the amount of pain
experienced by a patient are well known to those skilled in the
art. For example, a patient can be given a pain assessment test in
which the patient quantifies the degree of pain based on a scale.
One example would be assigning the patient's pain a number based on
a scale of 1 to 10, where a "10" would indicate the worst degree of
pain the patient might imagine. A pain measure of 4 from an
original pain score of 8 would be a 50% reduction in pain. Thus,
the amount of conjugate required to achieve that 50% reduction in
pain could be considered 1 U of the clostridial toxin
component-targeting moiety conjugate. Alternatively, the patient's
pain may be measured as the duration of pain. One unit of the
conjugate of the invention would accordingly reduce the duration of
pain by 50%. In addition, a number of physiological measures, such
as heart rate, respiratory rate, blood pressure, and diaphoresis,
may be used alone or together with the methods described above, to
quantify the amount of the patient's pain.
[0102] In an initial treatment, a low dosage may be administered at
one site to determine the patient's sensitivity to, and tolerance
of, the agent. Additional administration of the same or different
dosages may be performed as necessary.
[0103] In one embodiment, an agent of the invention is administered
intramuscularly. However, for some indications, extramuscular
injection may be the most efficacious route of administration. Such
injection may, for example, be made subcutaneously or, preferably,
perivascularly (to produce infiltration of the agent into
innervated tissue). In one embodiment, the site for injection of
the agent is in or near the extramuscular regions.
[0104] The agents may be administered by, for example, injection
using a needle or by needleless injection. The injections may be
repeated as necessary.
[0105] In needleless injection delivery methods, microprojectile
drug particles may be coated with an agent 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
microprojectiles penetrate through epidermal and dermal cells, or
are deposited in these cells, the agent is released. Individual
layers of skin cells or underlying muscle cells may be targeted for
the microprojectiles.
[0106] A range for administration of an agent, such as a
LH.sub.N-substance P conjugate (or targeting moiety substantially
similar to substance P including substance P precursors, fragments
and analogs), so as to achieve an antinociceptive effect in the
patient treated may be from about 10.sup.-2 U/kg to about 100 U/kg,
for example between about 10.sup.-1 U/kg to about 10 U/kg. Dosage
for a particular agent may depend on factors such as the condition
to be treated and the method of administration. An appropriate
dosage in a given circumstance may readily be determined by a
physician of ordinary skill.
[0107] A range for administration of an agent, such as an a L
chain-substance P conjugate, (or targeting moiety substantially
similar to substance P including substance P precursors, fragments
and analogs) so as to achieve an antinociceptive effect in the
patient treated may be from about 10.sup.-2 U/kg to about 100 U/kg,
for example between about 10.sup.-1 U/kg to about 10 U/kg. Dosage
for a particular agent may depend on factors such as the condition
to be treated and the method of administration. An appropriate
dosage in a given circumstance may readily be determined by a
physician of ordinary skill.
[0108] Dosage for a particular agent may depend on factors such as
the condition to be treated and the method of administration. A
physician of ordinary skill may readily determine an appropriate
dosage in a given circumstance.
[0109] The invention having been fully described, examples
illustrating its practice are set forth below. These examples
should not, however, be considered to limit the scope of the
invention, which is defined by the appended claims.
EXAMPLES
Example 1
Methods for Determining Potency of Botulinum Toxin
Component-Targeting Moiety Conjugates
[0110] The traditional unit of measure for botulinum toxin potency
is the mouse LD.sub.50 unit. That is, one unit (1 U) of botulinum
toxin is the amount that kills 50% of a group of 18-20 gram female
Swiss-Webster mice.
[0111] The unit of measure for potency of a botulinum toxin
component-targeting moiety conjugate may also be determined by
LD.sub.50 assays. In particular, 1 U of the botulinum toxin
component-targeting moiety component conjugate (for example, a
LH.sub.N-substance P conjugate) is the amount of the conjugate that
kills 50% of a group of 18-20 gram female Swiss-Webster mice.
[0112] Alternatively, potency of botulinum toxin
component-targeting moiety component conjugate may be determined by
the amount of pain reduction in a patient induced by a measured
amount of conjugate. For example, the pain reduction in a patient
may be estimated to be 50% upon injection of a measured amount of a
conjugate into a site of inflammation. Thus, the potency can be
measured as the amount of conjugate that reduces a patient's pain
by 50%.
[0113] Methods for assessing or quantifying the amount of pain
experienced by a subject are well known to those skilled in the
art. For example, a subject can be given a pain assessment test in
which the subject quantifies the degree of pain based on a scale.
One example would be assigning the subject's pain a number based on
a scale of 1 to 10, where a "10" would indicate the worst degree of
pain the subject might imagine. A pain measure of 4 from an
original pain score of 8 would be a 50% reduction in pain. Thus,
the amount of conjugate required to achieve that 50% reduction in
pain could be considered 1 U of the botulinum toxin
component-targeting moiety component conjugate. Alternatively, the
subject's pain may be measured as the duration of pain. One unit of
the conjugate of the invention would accordingly reduce the
duration of pain by 50%. In addition, a number of physiological
measures, such as heart rate, respiratory rate, blood pressure, and
diaphoresis, may be used individually or in combination to quantify
a subject's pain. These procedures may also be used in combination
with the subjective methods described above, to quantify the amount
of the subject's pain.
Example 2
Treatment of Neurogenic Inflammation Pain
[0114] A patient, age 45, experiencing acute neurogenic
inflammation pain is treated by intravenous administration with
between about 0.1 U/kg and about 30 U/kg, (for example, about 4 U),
of an agent comprising an LH.sub.N botulinum toxin type A-substance
P conjugate. Within 1-7 days after agent administration the
patient's pain is substantially alleviated. The duration of pain
reduction is from about 2 to about 6 months.
Example 3
Treatment of Neurogenic Inflammation Pain
[0115] A patient, age 36, experiencing inflammation pain of
neurogenic origin is treated by direct administration to the site
of pain of between about 0.1 U/kg and about 30 U/kg, (for example,
from about 1 U to about 10 U), of an agent comprising a botulinum
toxin type A L chain-substance P conjugate. A physician of ordinary
skill may readily determine the dosage, site of injection, and
frequency of administration. Within 1-7 days the pain symptoms are
substantially alleviated. The duration of pain reduction is from
about 2 to about 6 months.
Example 4
Treatment of Neurogenic Inflammation Pain
[0116] A patient, age 45, experiencing acute neurogenic
inflammatory pain is treated by administration, for example by
injection directly to a site of pain with an amount of an agent
comprising botulinum toxin type A, B, C, D, E, F and/or G H.sub.N
and type A, B, C, D, E, F and/or G L chain-substance P, substance P
analog and/or substance P fragment conjugate that reduces pain in
the subject by about 50%. A physician of ordinary skill may readily
determine the dosage, method of administration, and frequency of
administration. Within 1-7 days after agent administration the
patient's pain is alleviated by about 50% (in particular, the
patient's pain score is originally a 6, and after treatment, the
patient scores his pain as a 3). The duration of pain reduction is
from about 2 to about 6 months.
Example 5
Treatment of Neurogenic Inflammation Pain
[0117] A patient, age 36, experiencing neurogenic inflammation pain
(score of 8) is treated by intravenous administration of about 1 U
to about 20 U of an agent comprising a botulinum toxin type A, B,
C, D, E, F and/or G L chain-substance P, substance P analog and/or
substance P fragment conjugate that reduces the pain in the subject
by about 80% (score of about 1 to about 2). A physician of ordinary
skill may readily determine the dosage and frequency of
administration. Within 1-7 days the pain symptoms are reduced by
about 80%. The duration of pain reduction is from about 2 to about
6 months.
Example 6
Treatment of Fibromyalgia
[0118] A 37-year old woman complains of pain "all over,"
specifically in the occiput, neck, shoulders, lower back, hips, and
right leg.
[0119] At the time of evaluation, the patient complains
specifically of occipital headache, which she describes as burning
and aching. She complains of neck pain and stiffness. Aching pain
in the upper back and shoulders is constant, as is pain in a
band-like fashion across the lower back. She also notes a great
increase in her upper back and neck pain. The patient injures her
lower back one year prior to this evaluation--while working as a
nurse's aide as she transfers a patient. She subsequently continues
to experience lower back, hip, and right leg pain and
stiffness.
[0120] Upon examination, it is found that the patient tests
positive for pain sensitivity in 11 of the 18 fibromyalgia tender
points. Specifically, tenderness is present in the left and right
occiput, the left and right cervical regions, the left and right
trapizious, the left and right gluteal, the left and right
supraspinatus and the right greater trochanter. A diagnosis of
fibromyalgia is made.
[0121] The patient is treated by injection of about 1 to about 40
units of botulinum toxin type A LH.sub.N-substance P conjugate into
tendon insertion sites near the perceived sources of pain. Within
one week of treatment, the patient notes a substantial decrease in
pain felt throughout her body. Pain relief lasts for approximately
eight months.
Example 7
Treatment of Myofascial Pain Syndrome
[0122] A physical examination reveals a 38-year old woman who
suffers nerve damage in an auto accident at the age of 28. She
suffers from a steady pain in addition to allodynia in her legs.
The patient walks with a normal gait and is able to perform heel
walk and toe walk without difficulty. Range of motion of the lumbar
and cervical spine is complete. Motor and sensory examination of
the upper and lower extremities reveals mild S1 sensory loss on the
right side. Reflexes are intact at the knees and ankles.
[0123] Radiographics of the cervical and lumbar spine are
essentially normal demonstrating only mild degenerative
changes.
[0124] The patient is diagnosed with myofacial pain syndrome and is
treated with injections of about 1 to about 60 units of botulinum
toxin type A L chain, type B H.sub.N hybrid LH.sub.N-substance P
conjugate. A physician of ordinary skill may readily determine the
dosage, method of administration, and frequency of administration.
After approximately 6 days the patient reports herself to be pain
free. The relief from pain lasts approximately 6 months at which
time the injections are repeated.
Example 8
Treatment of Irritable Bowel
[0125] A 30 year old man visits his family physician with chief
complaints of persistent pain in the upper and lower back,
indigestion, constant tiredness and frequent urination.
[0126] The man discloses bouts of gastrointestinal discomfort, both
before and after meals. The patient denies any drug use or
cigarette smoking, except for an occasional alcoholic drink. Visual
observation reveals a man not under distress. Cranial tests are
normal. Reflexes are normal. A blood chemistry is ordered. With an
exception of a slightly lower K+ level, all other values are
normal. Antinuclear antibody, rheumatoid factor, creatine kinase,
magnesium, lyme titre, and thyroid function tests are negative. A
diagnosis of irritable bowel syndrome is made. The physician
prescribes injections of about 1 to about 10 units of botulinum
toxin type A LH.sub.N-substance P conjugate into the wall of the
lower small intestine. After one week, the patient reports only
mild relief. A further injection is made using about 1 to about 40
units of the same conjugate agent. Pain relief of up to 80% is
realized one week after this treatment.
Example 9
Treatment of Migraine Headache
[0127] A 34-year-old woman seeks medical attention for migraine
headache. The patient reports the symptoms persisting for
approximately 5 years. In addition, the patient reports an
inability to obtain restful sleep. The patient has been prescribed
numerous medications over the years including tylenol III,
Propanolol hydrochloride, Dihydroergotamine mesylate, Naratriptan
hydrochloride, Sumatriptan succinate and Zolmitriptan. All have met
with moderate to little effect.
[0128] The patient is intravenously injected with about 1 to about
120 units of a botulinum toxin type A L chain, type B H.sub.N,
LH.sub.N-substance P conjugate.
[0129] At 1 week a physical examination reveals no sign of migraine
headache pain. Pain relief last for approximately 9 months.
Example 10
Treatment of Arthritis
[0130] A patient, age 45, complains of pain in the back and left
hip. The patient also reports symptoms of pain and lack of mobility
in his left wrist, and in several of his fingers. The patient is
diagnosed with rheumatoid arthritis. The patient is treated by an
injection of about 1 to about 50 units of a botulinum toxin type A
L chain, type B H.sub.N hybrid LH.sub.N-substance P conjugate and
or a botulinum toxin type A L chain-substance P conjugate into or
near the regions of pain. A physician of ordinary skill may readily
determine the specific dosage, site of injection, and frequency of
administration. Within 1-7 days after administration the patient's
pain is substantially alleviated. The duration of the pain
alleviation is from about 1 to about 6 months.
Example 11
Treatment of Interstitial Cystitis
[0131] Upon examination, a 56 year old woman complains of chronic
pelvic pain, pain with sexual relations, sleep difficulties and
incontinence. The patient also reports symptoms of irritable bowel
and fatigue. She states that she is suffering from these symptoms
for over a year and that just recently the pain is increasing
substantially. A diagnosis of interstitial cystitis is made. The
patient is treated by injection of about 2 to about 200 units of
botulinum toxin type A LH.sub.N-substance P conjugate into the
bladder wall. A physician of ordinary skill may readily determine
the specific dosage, site of injection, and frequency of
administration. 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 12
Treatment of Pain Associated with Fibromyalgia Tender Points
[0132] A 36 year old woman has a 15 year history of chronic pain in
the upper torso area. Fifteen years prior to evaluation she notes a
decrease in mobility in her left elbow and knees. The pain in the
left side of her body is thought to be worse than in the right.
Upon examination it is revealed that the patient is pain sensitive
to firm pressure that is applied sequentially to eight of the
eighteen fibromyalgia tender points. In order for a diagnosis of
fibromyalgia to be made, 11 of the 18 fibromyalgia tender points
must be pain sensitive to application of pressure. Since this
standard is not met, the patient is not diagnosed as having
fibromyalgia. She is treated with antidepressant medication
including amytriptyline (Elavil). The medication has little effect
on the level of pain experienced by the patient.
[0133] The patient is injected with about 1 to about 20 units of
botulinum toxin type A L chain-substance P conjugate into the sites
where the patient perceives the pain to originate. A physician of
ordinary skill may readily determine the specific dosage, site of
injection, and frequency of administration.
[0134] Several days after the injections she notes substantial
improvement in her pain. This gradually improves over a 2 to 3 week
period in which she notes increased mobility in her elbow and knee
joints. The patient states that the pain is better than at any time
in the last 4 years. The improved condition persists for up to 6
months.
Sequence CWU 1
1
18111PRTHomo sapiensMOD_RES(11)...(11)Xaa at position 11 is
methionine amide 1Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Xaa1 5
10212PRTHomo sapiens 2Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met
Gly1 5 10313PRTHomo sapiens 3Arg Pro Lys Pro Gln Gln Phe Phe Gly
Leu Met Gly Lys1 5 10414PRTHomo sapiens 4Arg Pro Lys Pro Gln Gln
Phe Phe Gly Leu Met Gly Lys Arg1 5 10512PRTArtificial
SequenceSynthetic Substance P 5Arg Pro Lys Pro Gln Gln Phe Phe Gly
Leu Met Xaa1 5 10613PRTArtificial SequenceSynthetic Substance P
6Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Xaa1 5
10714PRTArtificial SequenceSynthetic Substance P 7Arg Pro Lys Pro
Gln Gln Phe Phe Gly Leu Met Gly Lys Xaa1 5 10812PRTArtificial
SequenceSynthetic Substance P 8Arg Pro Lys Pro Gln Gln Phe Phe Gly
Leu Met Xaa1 5 10913PRTArtificial SequenceSynthetic Substance P
9Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met Gly Xaa1 5
101014PRTArtificial SequenceSynthetic Substance P 10Arg Pro Lys Pro
Gln Gln Phe Phe Gly Leu Met Gly Lys Xaa1 5 10114PRTHomo sapiens
11Arg Pro Lys Pro1127PRTHomo sapiens 12Arg Pro Lys Pro Gln Gln Phe1
5139PRTHomo sapiens 13Arg Pro Lys Pro Gln Gln Phe Phe Gly1
51411PRTArtificial SequenceSynthetic Substance P 14Arg Xaa Lys Pro
Gln Gln Xaa Phe Xaa Leu Xaa1 5 101512PRTArtificial
SequenceSynthetic Substance P 15Arg Xaa Lys Pro Gln Gln Xaa Phe Xaa
Leu Met Gly1 5 101611PRTArtificial SequenceSynthetic Substance P
16Arg Xaa Lys Pro Gln Gln Xaa Phe Xaa Leu Xaa1 5
101712PRTArtificial SequenceSynthetic Substance P 17Arg Xaa Lys Pro
Gln Gln Xaa Phe Xaa Leu Met Gly1 5 101811PRTArtificial
SequenceSynthetic Substance P 18Arg Pro Cys Pro Gln Cys Phe Tyr Gly
Pro Xaa1 5 10
* * * * *