U.S. patent application number 11/259514 was filed with the patent office on 2006-11-16 for methods of modulating intracellular degradation rates of toxins.
This patent application is currently assigned to ALLERGAN, INC.. Invention is credited to Kei Roger Aoki, Shengwen Li.
Application Number | 20060257430 11/259514 |
Document ID | / |
Family ID | 35506053 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257430 |
Kind Code |
A1 |
Li; Shengwen ; et
al. |
November 16, 2006 |
Methods of modulating intracellular degradation rates of toxins
Abstract
The present invention provides for methods of modulating the
degradation rate of a toxin in a cell, thereby modulating the
half-life of the toxin. Particularly, the invention features
methods of modulating the degradation rate of a toxin by modulating
fusion between a lysosome and an endosome that carries the toxin in
the cell.
Inventors: |
Li; Shengwen; (Irvine,
CA) ; Aoki; Kei Roger; (Irvine, CA) |
Correspondence
Address: |
ALLERGAN, INC., LEGAL DEPARTMENT
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
ALLERGAN, INC.
|
Family ID: |
35506053 |
Appl. No.: |
11/259514 |
Filed: |
October 25, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10880192 |
Jun 29, 2004 |
6991789 |
|
|
11259514 |
Oct 25, 2005 |
|
|
|
Current U.S.
Class: |
424/239.1 ;
424/94.63 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 21/02 20180101; A61K 9/0019 20130101; A61P 39/02 20180101;
A61K 38/4886 20130101; A61P 21/00 20180101 |
Class at
Publication: |
424/239.1 ;
424/094.63 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 39/08 20060101 A61K039/08 |
Claims
1. A method of modulating the degradation rate of a Clostridial
toxin in a cell, the method comprising the step of co-administering
to a cell the toxin and a lysosome-endosome fusion modulator.
2. The method according to claim 1, wherein the Clostridial toxin
is a beratti toxin, a butyricum toxin, a tetani toxin or a
botulinum toxin.
3. The method according to claim 2, wherein the botulinum toxin is
a botulinum toxin serotype A, botulinum toxin serotype B, botulinum
toxin serotype C1, botulinum toxin serotype D, botulinum toxin
serotype E, botulinum toxin serotype F or botulinum toxin serotype
G.
4. The method according to claim 1, wherein the lysosome-endosome
fusion modulator decreases the degradation rate of the Clostridial
toxin.
5. The method according to claim 1, wherein the lysosome-endosome
fusion modulator comprises a lysosome-endosome fusion
inhibitor.
6. The method according to claim 5, wherein the lysosome-endosome
fusion inhibitor is selected from the group consisting of a GTPase
inhibitor, ATPase inhibitor, brefeldin A, cytochalasin B,
Wortmannin, cytochalasin D, an inhibitor of actin filaments,
phorbol12-myristate 13-acetate (PMA), a stimulator of protein
kinase C, bafilomycin A, and mixtures of any of the following.
7. The method according to claim 6, wherein the GTPase inhibitor is
selected from the group consisting of a Rab GTPase inhibitor, a Rho
GTPase inhibitor, and mixtures of any of the following.
8. The method according to claim 6, wherein the ATPase inhibitor
comprises an ATPase associated with cellular activities (AAA) type
inhibitor.
9. The method according to claim 6, wherein the GTPase inhibitor is
selected from the group consisting of a guanine dissociation
inhibitor (GDI) protein, an isoprene binding domain of the guanine
dissociation inhibitor, a GTPase activating protein (GAP), a
fluoroaluminate (AIF.sub.4), a guanylyl 5-thiophosphate, a Y-27632
Rho kinase inhibitor, a C3 transferase, a Clostridium difficile
toxin A, a Clostridium difficile toxin B, a Clostridium sordellii
lethal toxin LT, a Escherichia coli cytotoxic necrotizing factor 1
(CNF1), a Escherichia coli cytotoxic necrotizing factor 2 (CNF2), a
Bordetella bronchiseptica dermonecrotizing toxin (DNT), and
mixtures of any of the following.
10. The method according to claim 1, wherein the lysosome-endosome
fusion modulator increases the degradation rate of the Clostridial
toxin.
11. The method according to claim 1, wherein the lysosome-endosome
fusion modulator comprises a fusion facilitator.
12. The method according to claim 11, wherein the fusion
facilitator comprises a GTPase activator, a type III secreted
toxin, or mixtures thereof.
13. The method according to claim 12, wherein the GTPase activator
comprises a guanine nucleotide exchange factor (GEF) protein, a GEF
protein mimic, or mixtures thereof.
14. The method according to claim 12, wherein the type III secreted
toxin is a Salmonella typhimurium SopE, a Salmonella SptP, a
Yersinia pseudotuberculosis YopE, a Yersinia YopT or a Pseudomonas
aeruginosa ExoS.
15. A method of modulating a half-life of a Clostridial toxin in a
mammal, the method comprising the step of co-administering to the
mammal the Clostridial toxin and a lysosome-endosome fusion
modulator.
16. The method according to claim 15, wherein the Clostridial toxin
is a beratti toxin, a butyricum toxin, a tetani toxin or a
botulinum toxin.
17. The method according to claim 16, wherein the botulinum toxin
is a botulinum toxin serotype A, botulinum toxin serotype B,
botulinum toxin serotype C1, botulinum toxin serotype D, botulinum
toxin serotype E, botulinum toxin serotype F or botulinum toxin
serotype G.
18. The method according to claim 15, wherein the lysosome-endosome
fusion modulator increases the half-life of the Clostridial
toxin.
19. The method according to claim 15, wherein the lysosome-endosome
fusion modulator comprises a lysosome-endosome fusion
inhibitor.
20. The method according to claim 19, wherein the lysosome-endosome
fusion inhibitor is selected from the group consisting of a GTPase
inhibitor, an ATPase inhibitor, brefeldin A, cytochalasin B,
Wortmannin, cytochalasin D, an inhibitor of actin filaments,
phorbol 12-myristate 13-acetate (PMA), a stimulator of protein
kinase C, bafilomycin A, and mixtures of any of the following.
21. The method according to claim 20, wherein the GTPase inhibitor
is selected from the group consisting of a Rab GTPase inhibitor, a
Rho GTPase inhibitor, and mixtures of any of the following.
22. The method according to claim 20, wherein the ATPase inhibitor
comprises an ATPase associated with cellular activities (AAA) type
inhibitor.
23. The method according to claim 20, wherein the GTPase inhibitor
is selected from the group consisting of a guanine dissociation
inhibitor (GDI) protein, an isoprene binding domain of the guanine
dissociation inhibitor, a GTPase activating protein (GAP), a
fluoroaluminate (AIF.sub.4), a guanylyl 5-thiophosphate, a Y-27632
Rho kinase inhibitor, a C3 transferase, a Clostridium difficile
toxin A, a Clostridium difficile toxin B, a Clostridium. sordellii
lethal toxin LT, a Escherichia coli cytotoxic necrotizing factor 1
(CNF1), a Escherichia coli cytotoxic necrotizing factor 2 (CNF2), a
Bordetella bronchiseptica dermonecrotizing toxin (DNT), and
mixtures of any of the following
24. The method according to claim 15, wherein the lysosome-endosome
fusion modulator decreases the half-life of the Clostridial
toxin.
25. The method according to claim 15, wherein the lysosome-endosome
fusion modulator comprises a lysosome-endosome facilitator.
26. The method according to claim 25, wherein the lysosome-endosome
facilitator comprises a GTPase activator, a type III secreted
toxin, or a mixture thereof.
27. The method according to claim 26, wherein the GTPase activator
comprises a guanine nucleotide exchange factor (GEF) protein, a GEF
protein mimic, or mixtures thereof.
28. The method according to claim 26, wherein the type III secreted
toxin is a Salmonella typhimurium SopE, a Salmonella SptP, a
Yersinia pseudotuberculosis YopE, a Yersinia YopT or a Pseudomonas
aeruginosa ExoS.
29. A method of treating a biological disorder in a patient, the
method comprising the step of co-administering to a patient in need
thereof a Clostridial toxin and a lysosome-endosome fusion
inhibitor modulator.
30. The method according to claim 29, wherein the biological
disorder comprises at least one of a neuromuscular disorder, an
autonomic disorder and pain.
31. The method according to claim 29, wherein the Clostridial toxin
is a beratti toxin, a butyricum toxin, a tetani toxin or a
botulinum toxin.
32. The method according to claim 31, wherein the botulinum toxin
is a botulinum toxin serotype A, botulinum toxin serotype B,
botulinum toxin serotype C1, botulinum toxin serotype D, botulinum
toxin serotype E, botulinum toxin serotype F or botulinum toxin
serotype G.
33. The method according to claim 29, wherein the lysosome-endosome
fusion modulator decreases the degradation rate of the Clostridial
toxin.
34. The method according to claim 29, wherein the lysosome-endosome
fusion modulator increases the half-life of the Clostridial
toxin.
35. The method according to claim 29, wherein the lysosome-endosome
fusion modulator comprises a lysosome-endosome fusion
inhibitor.
36. The method according to claim 35, wherein the lysosome-endosome
fusion inhibitor is selected from the group consisting of a GTPase
inhibitor, ATPase inhibitor, brefeldin A, cytochalasin B,
Wortmannin, cytochalasin D, an inhibitor of actin filaments,
phorbol12-myristate 13-acetate (PMA), a stimulator of protein
kinase C, bafilomycin A, and mixtures of any of the following.
37. The method according to claim 36, wherein the GTPase inhibitor
is selected from the group consisting of a Rab GTPase inhibitor, a
Rho GTPase inhibitor, and mixtures of any of the following.
38. The method according to claim 36, wherein the ATPase inhibitor
comprises an ATPase associated with cellular activities (AAA) type
inhibitor.
39. The method according to claim 36, wherein the GTPase inhibitor
is selected from the group consisting of a guanine dissociation
inhibitor (GDI) protein, an isoprene binding domain of the guanine
dissociation inhibitor, a GTPase activating protein (GAP), a
fluoroaluminate (AIF.sub.4), a guanylyl 5-thiophosphate, a Y-27632
Rho kinase inhibitor, a C3 transferase, a Clostridium difficile
toxin A, a Clostridium difficile toxin B, a Clostridium sordellii
lethal toxin LT, a Escherichia coli cytotoxic necrotizing factor 1
(CNF1), a Escherichia coli cytotoxic necrotizing factor 2 (CNF2), a
Bordetella bronchiseptica dermonecrotizing toxin (DNT), and
mixtures of any of the following
40. The method according to claim 29, wherein the lysosome-endosome
fusion modulator increases the degradation rate of the Clostridial
toxin.
41. The method according to claim 29, wherein the lysosome-endosome
fusion modulator decreases the half-life of the Clostridial
toxin.
42. The method according to claim 29, wherein the lysosome-endosome
fusion modulator comprises a fusion facilitator.
43. The method according to claim 42, wherein the fusion
facilitator comprises a GTPase activator, a type III secreted
toxin, or mixtures thereof.
44. The method according to claim 43, wherein the GTPase activator
comprises a guanine nucleotide exchange factor (GEF) protein, a GEF
protein mimic, or mixtures thereof.
45. The method according to claim 43, wherein the type III secreted
toxin is a Salmonella typhimurium SopE, a Salmonella SptP, a
Yersinia pseudotuberculosis YopE, a Yersinia YopT or a Pseudomonas
aeruginosa ExoS.
46. (canceled)
Description
[0001] This continuation patent application claims priority
pursuant to 35 U.S.C. .sctn. 120 to U.S. non-provisional patent
application Ser. No. 10/880,192 filed Jun. 29, 2004, which is
hereby incorporated by reference in its entirety.
FIELD OF INVENTION
[0002] This invention broadly relates to intracellular trafficking.
Particularly, the invention relates to methods of modulating the
degradation rates of toxins in a cell.
BACKGROUND
[0003] The genus Clostridium has more than one hundred and twenty
seven species, grouped according to their morphology and functions.
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 shows a high
affinity for cholinergic motor neurons. Symptoms of botulinum toxin
intoxication can progress from difficulty walking, swallowing, and
speaking to paralysis of the respiratory muscles and death.
[0004] Botulinum toxin type A is the most lethal natural biological
agent known to man. About 50 picograms of a commercially available
botulinum toxin type A (purified neurotoxin complex, available from
Allergan, Inc., of Irvine, Calif. under the trade name BOTOX.RTM.
in 100 unit vials) is a LD50 in mice (i.e. 1 unit). One unit of
BOTOX.RTM. contains about 50 picograms (about 56 attomoles) of
botulinum toxin type A complex. Interestingly, on a molar basis,
botulinum toxin type A is about 1.8 billion times more lethal than
diphtheria, about 600 million times more lethal than sodium
cyanide, about 30 million times more lethal than cobra toxin and
about 12 million times more lethal than cholera. Singh, Critical
Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) of
Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New
York (1976) (where the stated LD50 of botulinum toxin type A of 0.3
ng equals 1 U is corrected for the fact that about 0.05 ng of
BOTOX.RTM. equals 1 unit). One unit (U) of botulinum toxin is
defined as the LD50 upon intraperitoneal injection into female
Swiss Webster mice weighing 18 to 20 grams each.
[0005] Seven generally immunologically distinct botulinum toxins
have been characterized, these being respectively botulinum toxin
serotypes A, B, C.sub.1, D, E, F and G each of which is
distinguished by neutralization with type-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 botulinum
toxin type A is 500 times more potent, as measured by the rate of
paralysis produced in the rat, than is botulinum toxin type B.
Additionally, botulinum toxin type B has been determined to be
non-toxic in primates at a dose of 480 U/kg which is about 12 times
the primate LD50 for botulinum toxin type A. Moyer E et al.,
Botulinum Toxin Type B: Experimental and Clinical Experience, being
chapter 6, pages 71-85 of "Therapy with Botulinum Toxin", edited by
Jankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxin
apparently binds with high affinity to cholinergic motor neurons,
is translocated into the neuron and blocks the release of
acetylcholine. Additional uptake can take place through low
affinity receptors, as well as by phagocytosis and pinocytosis.
[0006] 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 heavy chain (the H chain or HC) and a cell
surface receptor. The receptor is thought to be different for each
type of botulinum toxin and for tetanus toxin. The carboxyl end
segment of the HC appears to be important for targeting of the
botulinum toxin to the cell surface.
[0007] In the second step, the botulinum toxin crosses the plasma
membrane of the target cell. The botulinum toxin is first engulfed
by the cell through receptor-mediated endocytosis, fused with an
endosome and an endosome containing the botulinum toxin is formed.
The toxin then escapes the endosome into the cytoplasm of the cell.
This step is thought to be mediated by the amino end segment of the
HC, the 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 botulinum toxin to embed itself in the endosomal
membrane. The botulinum toxin (or at least the light chain of the
botulinum) then translocates through the endosomal membrane into
the cytoplasm.
[0008] The last step of the mechanism of botulinum toxin activity
appears to involve reduction of the disulfide bond joining the
heavy chain, H chain, and the light chain, 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 types 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 cytoplasmic surface of the synaptic vesicle is removed as a
result of any one of these cleavage events. Botulinum toxin
serotype A and E cleave SNAP-25. Botulinum toxin serotype C1 was
originally thought to cleave syntaxin, but was found to cleave
syntaxin and SNAP-25. Each of the botulinum toxins specifically
cleaves a different bond, except botulinum toxin type B (and
tetanus toxin) which cleave the same bond. Each of these cleavages
block the process of vesicle-membrane docking, thereby preventing
exocytosis of vesicle content.
[0009] Botulinum toxins have been used in clinical settings for the
treatment of neuromuscular disorders characterized by hyperactive
skeletal muscles (i.e. motor disorders). In 1989 a botulinum toxin
type A complex was approved by the U.S. Food and Drug
Administration for the treatment of blepharospasm, strabismus and
hemifacial spasm. Subsequently, a botulinum toxin type A was also
approved by the FDA for the treatment of cervical dystonia and for
the treatment of glabellar lines, and a botulinum toxin type B was
approved for the treatment of cervical dystonia. Non-type A
botulinum toxin serotypes apparently have a lower potency and/or a
shorter duration of activity as compared to botulinum toxin type A.
Clinical effects of peripheral intramuscular botulinum toxin type A
are usually seen within one week of injection. The typical duration
of symptomatic relief from a single intramuscular injection of
botulinum toxin type A averages about three months, although
significantly longer periods of therapeutic activity have been
reported.
[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 types A and E both cleave the 25
kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they
target different amino acid sequences within this protein.
Botulinum toxin types 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
type C1 has been shown to cleave both syntaxin and SNAP-25. These
differences in mechanism of action may affect the relative potency
and/or duration of action of the various botulinum toxin serotypes.
Apparently, a substrate for a botulinum toxin can be found in a
variety of different cell types. See e.g. Biochem J 1;339 (pt
1):159-65:1999, and Mov Disord, 10(3):376:1995 (pancreatic islet B
cells contains at least SNAP-25 and synaptobrevin).
[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 botulinum toxin type A complex can be produced by
Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum
toxin types B and C1 is apparently produced as only a 700 kD or 500
kD complex. Botulinum toxin type D is produced as both 300 kD and
500 kD complexes. Finally, botulinum toxin types 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 proteins 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 a botulinum 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
(Habermann E., et al., Tetanus Toxin and Botulinum A and C
Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse
Brain, J Neurochem 51(2);522-527:1988) CGRP, substance P and
glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks
Glutamate Exocytosis From Guinea Pig Cerebral Cortical
Synaptosomes, Eur J. Biochem 165;675-681:1897. Thus, when adequate
concentrations are used, stimulus-evoked release of most
neurotransmitters can be blocked by botulinum toxin. See e.g.
Pearce, L. B., Pharmacologic Characterization of Botulinum Toxin
For Basic Science and Medicine, Toxicon 35(9);1373-1412 at 1393;
Bigalke H., et al., Botulinum A Neurotoxin Inhibits Non-Cholinergic
Synaptic Transmission in Mouse Spinal Cord Neurons in Culture,
Brain Research 360;318-324:1985; Habermann E., Inhibition by
Tetanus and Botulinum A Toxin of the release of [3H]Noradrenaline
and [3H]GABA From Rat Brain Homogenate, Experientia
44;224-226:1988, Bigalke H., et al., Tetanus Toxin and Botulinum A
Toxin Inhibit Release and Uptake of Various Transmitters, as
Studied with Particulate Preparations From Rat Brain and Spinal
Cord, Naunyn-Schmiedeberg's Arch Pharmacol 316;244-251:1981, and;
Jankovic J. et al., Therapy With Botulinum Toxin, Marcel Dekker,
Inc., (1994), page 5.
[0013] Botulinum toxin type 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 C1, 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 botulinum toxin
type 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
botulinum toxin type B toxin is likely to be inactive, possibly
accounting for the known significantly lower potency of botulinum
toxin type B as compared to botulinum toxin type 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 botulinum
toxin type B has, upon intramuscular injection, a shorter duration
of activity and is also less potent than botulinum toxin type A at
the same dose level.
[0014] High quality crystalline botulinum toxin type A can be
produced from the Hall A strain of Clostridium botulinum with
characteristics of .gtoreq.3.times.107 U/mg, an A260/A278 of less
than 0.60 and a distinct pattern of banding on gel electrophoresis.
The known Shantz process can be used to obtain crystalline
botulinum toxin type A, as set forth in Shantz, E. J., et al,
Properties and use of Botulinum toxin and Other Microbial
Neurotoxins in Medicine, Microbiol Rev. 56;80-99:1992. Generally,
the botulinum toxin type A complex can be isolated and purified
from an anaerobic fermentation by cultivating Clostridium botulinum
type A in a suitable medium. The known process can also be used,
upon separation out of the non-toxin proteins, to obtain pure
botulinum toxins, such as for example: purified botulinum toxin
type A with an approximately 150 kD molecular weight with a
specific potency of 1-2.times.10.sup.8 LD50 U/mg or greater;
purified botulinum toxin type B with an approximately 156 kD
molecular weight with a specific potency of 1-2.times.10.sup.8 LD50
U/mg or greater, and; purified botulinum toxin type F with an
approximately 155 kD molecular weight with a specific potency of
1-2.times.10.sup.7 LD50 U/mg or greater.
[0015] Botulinum toxins and/or botulinum toxin complexes can be
obtained from List Biological Laboratories, Inc., Campbell, Calif.;
the Centre for Applied Microbiology and Research, Porton Down ,
U.K.; Wako (Osaka, Japan), Metabiologics (Madison, Wis.) as well as
from Sigma Chemicals of St Louis, Mo. Pure botulinum toxin can also
be used to prepare a pharmaceutical compound.
[0016] As with enzymes generally, the biological activities of the
botulinum toxins (which are intracellular peptidases) is dependent,
at least in part, upon their three dimensional conformation. Thus,
botulinum toxin type A is detoxified by heat, various chemicals
surface stretching and surface drying. Additionally, it is known
that dilution of a botulinum toxin complex obtained by the known
culturing, fermentation and purification to the much, much lower
toxin concentrations used for pharmaceutical compound formulation
results in rapid detoxification of the toxin unless a suitable
stabilizing agent is present. Dilution of the toxin from milligram
quantities to a solution containing nanograms per milliliter
presents significant difficulties because of the rapid loss of
specific toxicity upon such great dilution. Since the botulinum
toxin may be used months or years after the toxin containing
pharmaceutical compound is formulated, the toxin can be stabilized
with a stabilizing agent such as albumin and gelatin.
[0017] A commercially available botulinum toxin containing
pharmaceutical compound is sold under the trademark BOTOX.RTM.
(available from Allergan, Inc., of Irvine, Calif.). BOTOX.RTM.
consists of a purified botulinum toxin type A complex, albumin and
sodium chloride packaged in sterile, vacuum-dried form. The
botulinum toxin type A is made from a culture of the Hall strain of
Clostridium botulinum grown in a medium containing N-Z amine and
yeast extract. The botulinum toxin type A complex is purified from
the culture solution by a series of acid precipitations to a
crystalline complex consisting of the active high molecular weight
toxin protein and an associated hemagglutinin protein. The
crystalline complex is re-dissolved in a solution containing saline
and albumin and sterile filtered (0.2 microns) prior to
vacuum-drying. The vacuum-dried product is stored in a freezer at
or below -5.degree. C. BOTOX.RTM. can be reconstituted with
sterile, non-preserved saline prior to intramuscular injection.
Each vial of BOTOX.RTM. contains about 100 units (U) of Clostridium
botulinum toxin type A purified neurotoxin complex, 0.5 milligrams
of human serum albumin and 0.9 milligrams of sodium chloride in a
sterile, vacuum-dried form without a preservative.
[0018] To reconstitute vacuum-dried BOTOX.RTM., sterile normal
saline without a preservative; (0.9% Sodium Chloride Injection) is
used by drawing up the proper amount of diluent in the appropriate
size syringe. Since BOTOX.RTM. may be denatured by bubbling or
similar violent agitation, the diluent is gently injected into the
vial. For sterility reasons BOTOX.RTM. is preferably administered
within four hours after the vial is removed from the freezer and
reconstituted. During these four hours, reconstituted BOTOX.RTM.
can be stored in a refrigerator at about 2.degree. C. to about
8.degree. C. Reconstituted, refrigerated BOTOX.RTM. has been
reported to retain its potency for at least about two weeks.
Neurology, 48:249-53:1997.
[0019] It has been reported that botulinum toxin type A has been
used in clinical settings as follows: [0020] (1) about 75-125 units
of BOTOX.RTM. per intramuscular injection (multiple muscles) to
treat cervical dystonia; [0021] (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 supercihii
muscle); [0022] (3) about 30-80 units of BOTOX.RTM. to treat
constipation by intrasphincter injection of the puborectalis
muscle; [0023] (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. [0024] (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). [0025] (6) to treat upper limb
spasticity following stroke by intramuscular injections of
BOTOX.RTM. into five different upper limb flexor muscles, as
follows: [0026] (a) flexor digitorum profundus: 7.5 U to 30 U
[0027] (b) flexor digitorum sublimus: 7.5 U to 30 U [0028] (c)
flexor carpi ulnaris: 10 U to 40 U [0029] (d) flexor carpi
radialis: 15 U to 60 U [0030] (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. [0031] (7) to treat migraine,
pericranial injected (injected symmetrically into glabellar,
frontalis and temporalis muscles) injection of 25 U of BOTOX.RTM.
has showed significant benefit as a prophylactic treatment of
migraine compared to vehicle as measured by decreased measures of
migraine frequency, maximal severity, associated vomiting and acute
medication use over the three month period following the 25 U
injection.
[0032] It is known that botulinum toxin type A can have an efficacy
for up to 12 months (European J. Neurology 6 (Supp 4):
S111-S1150:1999), and in some circumstances for as long as 27
months, when used to treat glands, such as in the treatment of
hyperhydrosis . See e.g. Bushara K., Botulinum toxin and
rhinorrhea, Otolaryngol Head Neck Surg 1996;114(3):507, and The
Laryngoscope 109:1344-1346:1999. However, the usual duration of an
intramuscular injection of BOTOX.RTM. is typically about 3 to 4
months.
[0033] The success of botulinum toxin type A to treat a variety of
clinical conditions has led to interest in other botulinum toxin
serotypes. Two commercially available botulinum type A preparations
for use in humans are BOTOX.RTM. available from Allergan, Inc., of
Irvine, Calif., and Dysport.RTM. available from Beaufour Ipsen,
Porton Down, England. A botulinum toxin type B preparation
(MyoBloc.RTM.) is available from Elan Pharmaceuticals of San
Francisco, Calif.
[0034] In addition to having pharmacologic actions at the
peripheral location, botulinum toxins may also have inhibitory
effects in the central nervous system. Work by Weigand et al,
Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292, 161-165, and
Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56
showed that botulinum toxin is able to ascend to the spinal area by
retrograde transport. As such, a botulinum toxin injected at a
peripheral location, for example intramuscularly, may be retrograde
transported to the spinal cord.
[0035] U.S. Pat. No. 5,989,545 discloses that a modified
clostridial neurotoxin or fragment thereof, preferably a botulinum
toxin, chemically conjugated or recombinantly fused to a particular
targeting moiety can be used to treat pain by administration of the
agent to the spinal cord.
[0036] It has been reported that use of a botulinum toxin to treat
various spasmodic muscle conditions can result in reduced
depression and anxiety, as the muscle spasm is reduced. Murry T.,
et al., Spasmodic dysphonia; emotional status and botulinum toxin
treatment, Arch Otolaryngol 1994 March; 120(3): 310-316; Jahanshahi
M., et al., Psychological functioning before and after treatment of
torticollis with botulinum toxin, J Neurol Neurosurg Psychiatry
1992; 55(3): 229-231. Additionally, German patent application DE
101 50 415 A1 discusses intramuscular injection of a botulinum
toxin to treat depression and related affective disorders.
[0037] A botulinum toxin has also been proposed for or has been
used to treat skin wounds (U.S. Pat. No. 6,447,787), various
autonomic nerve dysfunctions (U.S. Pat. No. 5,766,605), tension
headache, (U.S. Pat. No. 6,458,365), migraine headache pain (U.S.
Pat. No. 5,714,468), sinus headache (U.S. patent application Ser.
No. 429,069), post-operative pain and visceral pain (U.S. Pat. No.
6,464,986), neuralgia pain (U.S. patent application Ser. No.
630,587), hair growth and hair retention (U.S. Pat. No. 6,299,893),
dental related ailments (U.S. provisional patent application Ser.
No. 60/418,789), fibromyalgia (U.S. Pat. No. 6,623,742), various
skin disorders (U.S. patent application Ser. No. 10/731,973),
motion sickness (U.S. patent application Ser. No. 752,869),
psoriasis and dermatitis (U.S. Pat. No. 5,670,484), injured muscles
(U.S. Pat. No. 6,423,319) various cancers (U.S. Pat. No.
6,139,845), smooth muscle disorders (U.S. Pat. No. 5,437,291), down
turned mouth corners (U.S. Pat. No. 6,358,917), nerve entrapment
syndromes (U.S. patent application 2003 0224019), various impulse
disorders (U.S. patent application Ser. No. 423,380), acne (WO
03/011333) and neurogenic inflammation (U.S. Pat. No. 6,063,768).
Controlled release toxin implants are known (see e.g. U.S. Pat.
Nos. 6,306,423 and 6,312,708) as is transdermal botulinum toxin
administration (U.S. patent application Ser. No. 10/194,805).
[0038] Botulinum toxin type A has been used to treat epilepsia
partialis continua, a type of focal motor epilepsy. Bhattacharya
K., et al., Novel uses of botulinum toxin type A: two case reports,
Mov Disord 2000; 15(Suppl 2):51-52.
[0039] It is known that a botulinum toxin can be used to: weaken
the chewing or biting muscle of the mouth so that self inflicted
wounds and resulting ulcers can heal (Payne M., et al, Botulinum
toxin as a novel treatment for self mutilation in Lesch-Nyhan
syndrome, Ann Neurol 2002 September;52(3 Supp 1):S157); permit
healing of benign cystic lesions or tumors (Blugerman G., et al.,
Multiple eccrine hidrocystomas: A new therapeutic option with
botulinum toxin, Dermatol Surg 2003 May;29(5):557-9); treat anal
fissure (Jost W., Ten years' experience with botulinum toxin in
anal fissure, Int J Colorectal Dis 2002 September;17(5):298-302,
and; treat certain types of atopic dermatitis (Heckmann M., et al.,
Botulinum toxin type A injection in the treatment of lichen
simplex: An open pilot study, J Am Acad Dermatol 2002
April;46(4):617-9).
[0040] Additionally, a botulinum toxin may have an effect to reduce
induced inflammatory pain in a rat formalin model. Aoki K., et al,
Mechanisms of the antinociceptive effect of subcutaneous Botox:
Inhibition of peripheral and central nociceptive processing,
Cephalalgia 2003 September;23(7):649. Furthermore, it has been
reported that botulinum toxin nerve blockage can cause a reduction
of epidermal thickness. Li Y, et al., Sensory and motor denervation
influences epidermal thickness in rat foot glabrous skin, Exp
Neurol 1997;147:452-462 (see page 459). Finally, it is known to
administer a botulinum toxin to the foot to treat excessive foot
sweating (Katsambas A., et al., Cutaneous diseases of the foot:
Unapproved treatments, Clin Dermatol 2002
November-December;20(6):689-699; Sevim, S., et al., Botulinum
toxin-A therapy for palmar and plantar hyperhidrosis, Acta Neurol
Belg 2002 December;102(4):167-70), spastic toes (Suputtitada, A.,
Local botulinum toxin type A injections in the treatment of spastic
toes, Am J Phys Med Rehabil 2002 October;81(10):770-5), idiopathic
toe walking (Tacks, L., et al., Idiopathic toe walking: Treatment
with botulinum toxin A injection, Dev Med Child Neurol
2002;44(Suppl 91):6), and foot dystonia (Rogers J., et al.,
Injections of botulinum toxin A in foot dystonia, Neurology 1993
April;43(4 Suppl 2)).
[0041] Tetanus toxin, as wells as derivatives (i.e. with a
non-native targeting moiety), fragments, hybrids and chimeras
thereof can also have therapeutic utility. The tetanus toxin bears
many similarities to the botulinum toxins. Thus, both the tetanus
toxin and the botulinum toxins are polypeptides made by closely
related species of Clostridium (Clostridium tetani and Clostridium
botulinum, respectively). Additionally, both the tetanus toxin and
the botulinum toxins are dichain proteins composed of a light chain
(molecular weight about 50 kD) covalently bound by a single
disulfide bond to a heavy chain (molecular weight about 100 kD).
Hence, the molecular weight of tetanus toxin and of each of the
seven botulinum toxins (non-complexed) is about 150 kD.
Furthermore, for both the tetanus toxin and the botulinum toxins,
the light chain bears the domain which exhibits intracellular
biological (protease) activity, while the heavy chain comprises the
receptor binding (immunogenic) and cell membrane translocational
domains.
[0042] Further, both the tetanus toxin and the botulinum toxins
exhibit a high, specific affinity for gangliocide receptors on the
surface of presynaptic cholinergic neurons. Receptor mediated
endocytosis of tetanus toxin by peripheral cholinergic neurons
results in retrograde axonal transport, blocking of the release of
inhibitory neurotransmitters from central synapses and a spastic
paralysis. Contrarily, receptor mediated endocytosis of botulinum
toxin by peripheral cholinergic neurons results in little if any
retrograde transport, inhibition of acetylcholine exocytosis from
the intoxicated peripheral motor neurons and a flaccid
paralysis.
[0043] Finally, the tetanus toxin and the botulinum toxins resemble
each other in both biosynthesis and molecular architecture. Thus,
there is an overall 34% identity between the protein sequences of
tetanus toxin and botulinum toxin type A, and a sequence identity
as high as 62% for some functional domains. Binz T. et al., The
Complete Sequence of Botulinum Neurotoxin Type A and Comparison
with Other Clostridial Neurotoxins, J Biological Chemistry
265(16);9153-9158:1990.
Acetylcholine
[0044] Typically only a single type of small molecule
neurotransmitter is released by each type of neuron in the
mammalian nervous system, although there is evidence which suggests
that several neuromodulators can be released by the same neuron.
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 bag 1 fibers of the muscle
spindle fiber, 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 as 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 heart rate by the vagal nerve.
[0045] 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.
[0046] 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 adrenal medulla,
as well as within the autonomic ganglia, that is on the cell
surface of the postganglionic neuron at the synapse between the
preganglionic and postganglionic neurons of both the sympathetic
and parasympathetic systems. Nicotinic receptors are also found in
many nonautonomic nerve endings, for example in the membranes of
skeletal muscle fibers at the neuromuscular junction.
[0047] Acetylcholine is released from cholinergic neurons when
small, clear, intracellular vesicles fuse with the presynaptic
neuronal cell membrane. A wide variety of non-neuronal secretory
cells, such as, adrenal medulla (as well as the PC12 cell line) and
pancreatic islet cells release catecholamines and parathyroid
hormone, 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.
[0048] 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.
[0049] One of the reasons that BoNT/A has been selected over the
other serotypes, for example serotypes B, C.sub.1, D, E, F, and G,
for clinical use is that BoNT/A has a substantially longer lasting
therapeutic effect. In other words, the inhibitory effect of BoNT/A
is more persistent. Therefore, the other serotypes of botulinum
toxins could potentially be effectively used in a clinical
environment if their half-lives in the mammal are enhanced. For
example, parotid sialocele is a condition where the patient suffers
from excessive salivation. It is known that serotype D may be very
effective in reducing excessive salivation. However, the half-life
of serotype D botulinum toxin is relatively short and thus may not
be practical for clinical use. If the half-life of serotype D may
be enhanced, it may effectively be used in a clinical environment
to treat, for example, parotid sialocele.
[0050] Another reason that BoNT/A has been a preferred neurotoxin
for clinical use is, as discussed above, its superb ability to
immobilize muscles through flaccid paralysis. For example, BoNT/A
is preferentially used to immobilize muscles and prevent limb
movements after a tendon surgery to facilitate recovery. However,
for some minor tendon surgeries, the healing time is relatively
short. It would be beneficial to be able to use BoNT/A without the
prolonged persistence for use in such circumstances so that the
patient can regain mobility at about the same time they recover
from the surgery. Thus, there is a need to have methods of
modulating the degradation rates or half-lives of neurotoxins.
SUMMARY OF THE INVENTION
[0051] The present invention provides for such unmet medical need
as described above. Accordingly, the present invention provides for
methods of modulating the degradation rate of a toxin in a cell. In
some embodiments, modulating the degradation rate of a toxin
comprises modulating fusion between a lysosome and an endosome that
carries the toxin in the cell. In some embodiments,
lysosome-endosome fusion modulators may be used. For example, a
lysosome-endosome fusion inhibitor may be used to inhibit the
fusion, and thereby decrease the degradation rate of the toxin in
the cell; and a lysosome-endosome fusion facilitator may be used to
facilitate the fusion, and thereby increase the degradation rate of
the toxin in the cell.
[0052] The present invention also features methods of modulating
the half-lives of toxins in a mammal. In some embodiments, the
methods comprise co-administering to the mammal a toxin with a
compound that modulates fusion of a lysosome and an endosome. The
present invention also provides for methods of treating a
biological disorder in a patient, for example, by co-administering
to a patient in need thereof a toxin and a lysosome-endosome fusion
inhibitor.
[0053] Any feature or combination of features described herein are
included within the scope of the present invention provided that
the features included in any such combination are not mutually
inconsistent as will be apparent from the context, this
specification, and the knowledge of one of ordinary skill in the
art. Additional advantages and aspects of the present invention are
apparent in the following detailed description and claims.
Definitions
[0054] "About" means approximately or nearly and in the context of
a numerical value or range set forth herein means .+-.10% of the
numerical value or range recited or claimed.
[0055] "Locally administering" means direct administration of a
pharmaceutic at or to the vicinity of a site on or within an animal
body, at which site a biological effect of the pharmaceutic is
desired. Local administration excludes systemic routes of
administration, such as intravenous or oral administration.
DESCRIPTION OF EMBODIMENTS
[0056] The present invention relates to methods of modulating the
degradation rate of a toxin in a cell. The invention is based, in
part, upon the discovery that modulating fusion between a lysosome
and an endosome that carries the toxin in the cell affects the
degradation rate of the toxin. Toxins that are within the scope of
the present invention include beratti toxin, butyricum toxin,
tetani toxin, BoNT/A, B, C.sub.1, D, E, F, and G. In some
embodiments, the present invention relates to methods of modulating
the degradation rate of a botulinum toxin in a cell.
[0057] Lysosomes are membrane bound organelles containing many
hydrolytic enzymes, which are optimally active at an acid pH. They
are distinguished from endosomes by the presence of the two
mannose-6-phosphate receptors (MPRs) and recycling cell surface
receptors. They are characteristically observed by electron
microscopy to be organelles of about 0.5 um diameter and often have
electron-dense cores. Lysosomes are often regarded as the terminal
degradation compartment of the endocytic pathway, and play
important roles in the degradation of phagocytosed material, in
autophagy, in crinophagy, and in the proteolysis of cyotsolic
proteins transported across the lysosomal membrane by a carrier
mediated mechanism.
[0058] As discussed above, toxins (e.g., botulinum toxins) are
endocytosed into a cell, and reside in an endosome therein. Without
wishing to limit the invention to any theory or mechanism of
operation, it is believed that toxins in the cells are subjected to
degradation when the endosome fuses with the lysosome. Further, it
is believed that lysosomes are able to fuse directly with late
endosomes to form hybrid organelles in which degradation of the
endocytosed material (e.g., a toxin such as a botulinum toxin)
takes place, and form which lysosomes are reformed.
[0059] The fusion between the lysosome and the endosome is ATP,
cytosol and temperature dependent. See Luzio J. P. et al., J. Cell
Science, 113:1515-1524 (2000), the disclosure of which is
incorporated in its entirety by reference herein. The fusion also
requires the presence of NSF (N-ethylmaleimide sensitive factor)
and SNAPs (soluble NSF attachment proteins) and is inhibited by
Rab-GDI (GDP dissociation inhibitor). This implies that Rab GTPase
is necessary for the fusion between the lysosome and the
endosome.
[0060] In some embodiments, the invention provides for a method of
decreasing (i.e., modulating) the degradation rate of a toxin in a
cell. In some embodiments, the method of decreasing the degradation
of a toxin in a cell comprises introducing a lysosome-endosome
fusion inhibitor into the cell to prevent the fusion of the
lysosome with the endosome. The lysosome-endosome fusion inhibitor
may be introduced into the cell through any means known by one of
ordinary skill in the art. For example, the lysosome-endosome
inhibitor may be introduced into the cell through the use of
electroporation techniques. Since the fusion between the lysosome
and the endosome may be dependent on GTP and ATP, a
lysosome-endosome fusion inhibitor may comprise a GTPase inhibitor,
an ATPase inhibitor or mixtures thereof. See Luzio J. P. et al., J.
Cell Science, 113: 1515-1524 (2000); and Fujita H. et al., J. Cell
Science, 116: 401-414 (2003), the disclosures of which are
incorporated in their entirety by reference herein. Other
lysosome-endosome fusion inhibitors include the serine-threonine
phosphatase inhibitor microcystin LR, mastoparan, and guanosine
5'-O(3'-thiotriphosphate) (Peters et al., Science, Vol 285, Issue
5430, 1084-1087, 13 Aug. 1999), Wortmannin (Biochem. J. (2003) 372
(861-869) (Printed in Great Britain)), brefeldin A (Golgi complex
disrupter), cytochalasin B (mircrofilament inhibitor), cytochalasin
D, an inhibitor of actin filaments, PMA (phorbol 12-myristate
13-acetate), a stimulator of protein kinase C, and bafilomycin A,
an inhibitor of lysosome/endosome function (Zuckers et al., Lab
Invest. 2002 December;82(12):1673-84; and Ramm et al., Hepatology,
1994 February;19(2):504-13.)
[0061] Intracellular membrane fusion can be divided into distinct
subreactions: priming, tethering and docking of the membranes, and
subsequent mixing of the bilayers and contents Most components
identified so far, such as NSF (NEM-sensitive fusion protein),
x-SNAP (soluble NSF attachment protein), SNAREs (SNAP receptors),
Rab-like guanosine triphosphatases (GTPases) and their cofactors,
and the LMA1 complex (low molecular weight activity), act in the
early steps of intracellular membrane fusion, mediating recognition
and association of the appropriate membranes. In contrast, there is
little information about the transition from docking to bilayer
mixing.
[0062] In some embodiments, GTPase inhibitors of the present
invention comprise a Rab GTPase inhibitor, a Rho GTPase inhibitor,
or a mixture thereof. In some embodiments, ATPase inhibitors of the
present invention comprise an ATPase associated with cellular
activities (AAA) type inhibitor. These inhibitors are commonly
known by one of ordinary skill in the art.
[0063] Non-limiting examples of GTPase inhibitors of the present
invention include a guanine dissociation inhibitor (GDI) protein,
an isoprene binding domain of the guanine dissociation inhibitor, a
GAP protein, an AIF.sub.4-, a guanylyl 5-thiophosphate, a Y-27632,
a C3 transferase, a Clostridium difficile toxin A, a Clostridium
difficile toxin B, a Clostridium. sordellii lethal toxin LT, a
Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a
Escherichia coli cytotoxic necrotizing factor 2 (CNF2) and a
Bordetella bronchiseptica dermonecrotizing toxin (DNT).
[0064] In some embodiments, the use of a lysosome-endosome fusion
inhibitor is effective to decrease the degradation rate of a toxin
(e.g., botulinum toxin) by about more than 10%. In some
embodiments, the use of a lysosome-endosome fusion inhibitor is
effective to decrease the degradation rate of a toxin (e.g.,
botulinum toxin) by about more than 25%. In some embodiments, the
use of a lysosome-endosome fusion inhibitor is effective to
decrease the degradation rate of a toxin (e.g., botulinum toxin) by
about more than 50%. In some embodiments, the use of a
lysosome-endosome fusion inhibitor is effective to decrease the
degradation rate of a toxin (e.g., botulinum toxin) by about more
than 100%.
[0065] In some embodiments, the invention provides for a method of
increasing (i.e., modulating) the degradation rate of a toxin,
e.g., a botulinum toxin, in a cell. In some embodiments, the method
of increasing the degradation of a botulinum toxin in a cell
comprises introducing a lysosome-endosome fusion facilitator into
the cell to enhance the fusion of the lysosome with the endosome.
In some embodiments, a fusion facilitator comprises a GTPase
activator, a type III secreted toxin, and ammonium chloride
(lysosome stablizers)
[0066] In some embodiments, the GTPase activator activator of the
present invention comprises a GEF protein, GEF protein mimic, or
mixtures thereof. In some embodiments, the type III secreted toxin
is a Salmonella typhimurium SopE, a Salmonella SptP, a Yersinia
pseudotubercolosis YopE, a Yersinia YopT or a Pseudomonas
aeruginosa ExoS.
[0067] In some embodiments, the use of a lysosome-endosome fusion
facilitator is effective to increase the degradation rate of a
toxin (e.g., botulinum toxin) by about more than 10%. In some
embodiments, the use of a lysosome-endosome fusion facilitator is
effective to increase the degradation rate of a toxin (e.g.,
botulinum toxin) by about more than 25%. In some embodiments, the
use of a lysosome-endosome fusion facilitator is effective to
increase the degradation rate of a toxin (e.g., botulinum toxin) by
about more than 50%. In some embodiments, the use of a
lysosome-endosome fusion facilitator is effective to increase the
degradation rate of a toxin (e.g., botulinum toxin) by about more
than 100%.
[0068] The present invention also features a method for modulating
the half-life of a botulinum toxin in a mammal. As used herein,
"half-life" refers to the time it takes for half of the toxin
population to be degraded in a mammal. In some embodiments, the
method comprises co-administering to the mammal a toxin with a
compound that modulates fusion of a lysosome and an endosome. As
used herein, "co-administering" includes sequential administration
of botulinum toxin followed by lysosome-endosome fusion modulator,
sequential administration of a lysosome-endosome fusion modulator
followed by a botulinum toxin, or simultaneous administration of a
botulinum toxin and a lysosome-endosome fusion modulator.
[0069] In some embodiments, the present invention provides for a
method of increasing (i.e., modulating) the half-life of the toxin
in a mammal. For example, the method of increasing the half-life of
the toxin comprises co-administering to the mammal the toxin and a
lysosome-endosome fusion inhibitor. In some embodiments, the
lysosome-endosome fusion inhibitor comprises a GTPase inhibitor, an
ATPase inhibitor, brefeldin A (Golgi complex disrupter),
cytochalasin B (mircrofilament inhibitor), Wortmannin, cytochalasin
D, an inhibitor of actin filaments, PMA, a stimulator of protein
kinase C, and bafilomycin A, an inhibitor of lysosome/endosome
function.
[0070] In some embodiments, the GTPase inhibitor comprises a
guanine dissociation inhibitor (GDI) protein, an isoprene binding
domain of the guanine dissociation inhibitor, a GAP protein, an
AIF.sub.4-, a guanylyl 5-thiophosphate, a Y-27632, a C3
transferase, a Clostridium difficile toxin A, a Clostridium
difficile toxin B, a Clostridium. sordellii lethal toxin LT, a
Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a
Escherichia coli cytotoxic necrotizing factor 2 (CNF2), a
Bordetella bronchiseptica dermonecrotizing toxin (DNT) or mixtures
thereof.
[0071] In some embodiments, the use of a lysosome-endosome fusion
inhibitor is effective to increase the half-life of a toxin (e.g.,
botulinum toxin) by about more than 10%. In some embodiments, the
use of a lysosome-endosome fusion inhibitor is effective to
increase the half-life of a toxin (e.g., botulinum toxin) by about
more than 25%. In some embodiments, the use of a lysosome-endosome
fusion inhibitor is effective to increase the half-life of a toxin
(e.g., botulinum toxin) by about more than 50%. In some
embodiments, the use of a lysosome-endosome fusion inhibitor is
effective to increase the half-life of a toxin (e.g., botulinum
toxin) by about more than 100%.
[0072] In some embodiments, the present invention provides a method
for decreasing (i.e., modulating) the half-life of the toxin in a
mammal. In some embodiments, the method of decreasing the half-life
comprises co-administering the mammal with the toxin and a
lysosome-endosome fusion facilitator.
[0073] In some embodiments, the lysosome-endosome facilitator
comprises a GTPase activator, a type III secreted toxin, or a
mixture thereof. In some embodiments, the GTPase activator
comprises a GEF protein, a GEF protein mimic or a mixture thereof.
In some embodiments, a type III secreted toxin is a Salmonella
typhimurium SopE, a Salmonella SptP, a Yersinia pseudotubercolosis
YopE, a Yersinia YopT or a Pseudomonas aeruginosa ExoS.
[0074] In some embodiments, the use of a lysosome-endosome fusion
facilitator is effective to decrease the half-life of a toxin
(e.g., botulinum toxin) by about more than 10%. In some
embodiments, the use of a lysosome-endosome fusion facilitator is
effective to decrease the half-life of a toxin (e.g., botulinum
toxin) by about more than 25%. In some embodiments, the use of a
lysosome-endosome fusion facilitator is effective to decrease the
half-life of a toxin (e.g., botulinum toxin) by about more than
50%. In some embodiments, the use of a lysosome-endosome fusion
facilitator is effective to decrease the half-life of a toxin
(e.g., botulinum toxin) by about more than 100%.
[0075] The present invention also features a toxin, e.g., a
botulinum toxin, fused with a fusion facilitator or fusion
inhibitor. The fusion may be carried out by conventional techniques
known in the art.
[0076] The present invention also provides for a method of treating
a biological disorder in a patient. In some embodiments, the method
comprises co-administering a botulinum toxin and a
lysosome-endosome fusion inhibitor to a patient in need thereof.
Non-limiting examples of biological disorder include a
neuromuscular disorder, an autonomic disorder and pain. The routes
of administration include, without limitation, transdermal,
peritoneal, subcutaneous, intramuscular, intravenous and
intrarectal.
[0077] In some embodiments, the method of treating a neuromuscular
disorder comprises locally co-administering a toxin and a
lysosome-endosome fusion inhibitor to a group of muscles.
[0078] In some embodiments, the method of treating an autonomic
disorder comprises locally administering a toxin and a
lysosome-endosome fusion inhibitor to a gland.
[0079] In some embodiments, the method of treating pain comprises
locally co-administering a toxin and a lysosome-endosome fusion
inhibitor to a site of pain. In some embodiments, the method of
treating pain comprises co-administering a toxin and a
lysosome-endosome fusion inhibitor to a spinal cord.
[0080] The present invention also provides methods for treating
toxin, e.g., botulinum toxin, intoxication in a mammal. In some
embodiments, the method comprises administering a lysosome-endosome
fusion facilitator to the mammal in need thereof, thereby treating
botulinum intoxication.
[0081] The doses of the toxin (e.g., botulinum toxin) and/or
lysosome-endosome fusion modulator to be administered depend on
many factors. One of ordinary skill will be able to readily
determine the specific dose for each specific compound.
[0082] Furthermore, the amount of the toxin and/or
lysosome-endosome fusion modulator 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. 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., 14th edition, published by McGraw Hill).
[0083] The toxins and/or lysosome-endosome fusion modulators of the
invention may be admixed, encapsulated, conjugated or otherwise
associated with other molecules or mixtures of compounds as, for
example, liposomes, formulations (oral, rectal, topical, etc.) for
assisting in uptake, distribution and/or absorption.
[0084] Pharmaceutical formulations for topical administration may
include transdermal patches, ointments, lotions, creams, gels,
drops, suppositories, sprays, liquids and powders. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners
and the like may be necessary or desirable. Coated condoms, gloves
and the like may also be useful. Preferred topical formulations
include those in which the compounds of the invention are in
admixture with a topical delivery agent such as lipids, liposomes,
fatty acids, fatty acid esters, steroids, chelating agents and
surfactants. Preferred lipids and liposomes (Chariot.TM. reagent)
include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). Compounds of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, compounds may be complexed to lipids, in particular
to cationic lipids. Preferred fatty acids and esters include but
are not limited arachidonic acid, oleic acid, eicosanoic acid,
lauric acid, caprylic acid, capric acid, myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S.
patent application Ser. No. 09/315,298 filed on May 20, 1999 which
is incorporated herein by reference in its entirety.
[0085] Formulations for oral administration include powders or
granules, microparticulates, nanoparticulates, suspensions or
solutions in water or non-aqueous media, capsules, gel capsules,
sachets, tablets or minitablets. Thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders may be desirable.
Preferred oral formulations are those in which compounds of the
invention are administered in conjunction with one or more
penetration enhancers surfactants and chelators. Preferred
surfactants include fatty acids and/or esters or salts thereof,
bile acids and/or salts thereof. Preferred bile acids/salts include
chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid
(UDCA), cholic acid, dehydrocholic acid, deoxycholic acid,
glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic
acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate
and sodium glycodihydrofusidate. Preferred fatty acids include
arachidonic acid, undecanoic acid, oleic acid, lauric acid,
caprylic acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable
salt thereof (e.g. sodium). Also preferred are combinations of
penetration enhancers, for example, fatty acids/salts in
combination with bile acids/salts. A particularly preferred
combination is the sodium salt of lauric acid, capric acid and
UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Compounds of the invention may be delivered orally, in granular
form including sprayed dried particles, or complexed to form micro
or nanoparticles. Compound complexing agents include poly-amino
acids; polyimines; polyacrylates; polyalkylacrylates,
polyoxethanes, polyalkylcyanoacrylates; cationized gelatins,
albumins, starches, acrylates, polyethyleneglycols (PEG) and
starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,
pollulans, celluloses and starches. Particularly preferred
complexing agents include chitosan, N-trimethylchitosan,
poly-L-lysine, polyhistidine, polyornithine, polyspermines,
protamine, polyvinylpyridine, polythiodiethylamino-methylethylene
P(TDAE), polyaminostyrene (e.g. p-amino),
poly(methylcyanoacrylate), poly(ethylcyanoacrylate),
poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),
poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG).
[0086] Formulations for parenteral, intrathecal or intraventricular
administration may include sterile aqueous solutions which may also
contain buffers, diluents and other suitable additives such as, but
not limited to, penetration enhancers, carrier compounds and other
pharmaceutically acceptable carriers or excipients.
[0087] Pharmaceutical compounds of the present invention include,
but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compounds may be generated
from a variety of components that include, but are not limited to,
preformed liquids, self-emulsifying solids and self-emulsifying
semisolids.
[0088] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0089] The compounds, e.g., toxins and/or lysosome-endosome fusion
modulator, of the present invention may be formulated into any of
many possible dosage forms such as, but not limited to, tablets,
capsules, gel capsules, liquid syrups, soft gels, suppositories,
and enemas. The compounds of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0090] In some embodiments of the present invention the
pharmaceutical compounds may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compounds and formulations is generally known
to those skilled in the pharmaceutical and formulation arts and may
be applied to the formulation of the compounds of the present
invention.
[0091] The following non-limiting examples provide those of
ordinary skill in the art with exemplary suitable methods for
practicing the present invention, and are not intended to limit the
scope of the invention.
EXAMPLE 1
Treatment of Pain Associated with Muscle Disorder.
[0092] A female patient is diagnosed as having temporomandibular
joint (TMJ) dysfunction with subluxation of the joint and is
treated with surgical orthoplasty meniscusectomy and condyle
resection. However, she continues to have difficulty with opening
and closing her jaw after the surgical procedures. The jaw
continues to exhibit considerable pain and immobility after these
surgical procedures. She is diagnosed as having post-surgical
myofascial pain syndrome and is injected with 7 U/kg of botulinum
toxin and a therapeutic amount of ATPase associated with cellular
activities (AAA) type inhibitor into the masseter and temporalis
muscles.
[0093] Several days after the injections she notes 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
improved condition persists for more than 27 months after the
original injection of the neurotoxin and the ATPase inhibitor.
EXAMPLE 2
Treatment of Excessive Sweating.
[0094] A 65 year old patient with excessive unilateral sweating is
treated by administering 0.05 U/kg to about 2 U/kg of a botulinum
toxin and a therapeutic amount of an ATPase inhibitor. The
administration is to the gland nerve plexus, ganglion, spinal cord
or central nervous system. The specific site of administration is
to be determined by the physician's knowledge of the anatomy and
physiology of the target glands and secretary cells. The cessation
of excessive sweating after the modified neurotoxin treatment is
more than 27 months.
EXAMPLE 3
Peripheral Administration of a Modified Neurotoxin to Treat
Nasopharyngeal Tumor Pain.
[0095] These tumors, most often squamous cell carcinomas, are
usually in the fossa of Rosenmuller and may invade the base of the
skull. Pain in the face is common. It is constant, dull-aching in
nature.
[0096] A 35 year old patient presents a nasopharyngeal tumor type
pain. Pain is found at the lower left cheek. The patient is treated
by a bolus injection of between about 0.05 U/kg to about 2 U/kg of
a botulinum toxin and a GTPase inhibitor intramuscularly to the
cheek. 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
more than 27 months.
EXAMPLE 4
Accidental Overdose in the Treatment of Postherpetic Neuralgia--Use
of Lysosome-Endosome Fusion Facilitator as an Antidote.
[0097] In an exemplary scenario, a 76 year old man presents a
postherpetic type pain. The pain is localized to the abdomen
region. The patient is treated by a bolus injection of between
about 0.05 U/kg to about 2 U/kg of a BOTOX.RTM. intradermally to
the abdomen. The treating physician accidentally administers an
excessive amount of BOTOX.RTM.. Upon realizing the error, the
physician administers to the same area a therapeutically effective
dose of a lysosome-endosome fusion facilitator. The particular dose
as well as the frequency of administrations the lysosome-endosome
fusion facilitator depend upon a variety of factors within the
skill of the treating physician. Within 1 day after BOTOX.RTM. and
corrective lysosome-endosome fusion facilitator administration, the
patient's pain is substantially alleviated.
[0098] Various articles and patents have been cited here. The
disclosures of these references are incorporated in their entirety
herein by reference herein.
[0099] 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.
* * * * *