U.S. patent application number 15/771635 was filed with the patent office on 2018-11-01 for injectable botulinum toxin formulations and methods of use thereof having long duration of therapeutic or cosmetic effect.
The applicant listed for this patent is Revance Therapeutics, Inc.. Invention is credited to Curtis L. Ruegg, Jacob M. Waugh.
Application Number | 20180311333 15/771635 |
Document ID | / |
Family ID | 58630886 |
Filed Date | 2018-11-01 |
United States Patent
Application |
20180311333 |
Kind Code |
A1 |
Ruegg; Curtis L. ; et
al. |
November 1, 2018 |
Injectable Botulinum Toxin Formulations And Methods Of Use Thereof
Having Long Duration Of Therapeutic Or Cosmetic Effect
Abstract
This invention provides novel injectable compositions comprising
botulinum toxin that may be administered to a subject for various
therapeutic, aesthetic and/or cosmetic purposes. The injectable
compositions embraced by the invention exhibit one or more
advantages over conventional botulinum toxin formulations,
including reduced antigenicity, a reduced tendency to undergo
unwanted localized diffusion following injection, increased
duration of clinical efficacy or enhanced potency relative, faster
onset of clinical efficacy, and/or improved stability. According to
the invention, single treatment of the compositions by injection
affords significant clinical responses and at least a 6-month
duration of effect in a subject undergoing treatment, as provided
by the described treatment methods.
Inventors: |
Ruegg; Curtis L.; (Redwood
City, CA) ; Waugh; Jacob M.; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Revance Therapeutics, Inc. |
Newark |
CA |
US |
|
|
Family ID: |
58630886 |
Appl. No.: |
15/771635 |
Filed: |
October 28, 2016 |
PCT Filed: |
October 28, 2016 |
PCT NO: |
PCT/US2016/059492 |
371 Date: |
April 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62248255 |
Oct 29, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/02 20180101;
A61P 25/04 20180101; A61P 25/00 20180101; A61K 8/64 20130101; A61P
1/00 20180101; A61P 17/02 20180101; A61P 37/08 20180101; C12Y
304/24069 20130101; A61K 2800/92 20130101; A61Q 19/08 20130101;
A61K 8/66 20130101; A61P 17/00 20180101; A61P 11/02 20180101; A61P
37/00 20180101; A61K 9/0021 20130101; A61P 13/10 20180101; A61P
27/02 20180101; A61P 9/14 20180101; A61P 17/10 20180101; A61K 39/08
20130101; A61P 19/02 20180101; A61P 25/14 20180101 |
International
Class: |
A61K 39/08 20060101
A61K039/08; A61Q 19/08 20060101 A61Q019/08; A61K 8/66 20060101
A61K008/66; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method of administering botulinum toxin to achieve an extended
duration therapeutic or cosmetic effect in an individual, the
method comprising: administering by injection a first treatment
dose of a sterile injectable composition into an area of the
individual in need of treatment to achieve the therapeutic or
cosmetic effect following a first treatment with the composition;
wherein the composition comprises a pharmaceutically acceptable
diluent suitable for injection; and a botulinum toxin component
selected from the group consisting of a botulinum toxin, a
botulinum toxin complex, or a reduced botulinum toxin complex; and
a positively charged carrier component comprising a positively
charged polylysine backbone having covalently attached thereto one
or more positively charged efficiency groups having an amino acid
sequence of (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; wherein the
botulinum toxin component is administered to the individual in a
treatment dose of 20 U to 60 U; wherein the positively charged
carrier is non-covalently associated with the botulinum toxin
component; and wherein the first treatment dose of the composition
administered by injection to the individual achieves the extended
duration therapeutic or cosmetic effect having at least about a 6
month to about a 10 month duration of effect, optionally, before a
second or subsequent treatment dose is administered.
2. A method of reducing wrinkles, lines, or furrows in an
individual in need thereof, the method comprising: administering to
the individual as a single dose injection a composition comprising:
a pharmaceutically acceptable diluent for injection; a botulinum
toxin component selected from the group consisting of a botulinum
toxin, a botulinum toxin complex, or a reduced botulinum toxin
complex; and a positively charged carrier component comprising a
positively charged polylysine backbone having covalently attached
thereto one or more positively charged efficiency groups having an
amino acid sequence of (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; wherein the
botulinum toxin is administered to the individual in an amount of
20 U to 60 U; wherein the positively charged carrier is
non-covalently associated with the botulinum component; and wherein
the single dose injection of the composition provides a single
treatment having at least about a six month to about a 10 month
duration of effect in reducing the wrinkles, lines, or furrows in
the individual, thereby extending treatment interval duration for
the individual.
3. A pharmaceutical composition in a sterile injectable formulation
for use in administering botulinum toxin to achieve an extended
duration therapeutic or cosmetic effect in an individual, said
composition comprising a pharmaceutically acceptable diluent
suitable for injection; a botulinum toxin component in a treatment
dose of 20 U to 60 U, wherein said botulinum toxin component is
selected from the group consisting of a botulinum toxin complex, a
reduced botulinum toxin complex, or a botulinum toxin; and a
positively charged carrier component comprising a positively
charged polylysine backbone having covalently attached thereto one
or more positively charged efficiency groups having an amino acid
sequence of (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; wherein the
positively charged carrier is non-covalently associated with the
botulinum toxin component; and wherein said treatment dose of the
composition achieves the extended duration therapeutic or cosmetic
effect having at least about a 6 month to about a 10 month duration
of effect in the individual administered said formulation by
injection.
4. A pharmaceutical composition in a sterile injectable formulation
for use in reducing wrinkles, lines, or furrows in an individual in
need thereof, said composition comprising: a botulinum toxin
component in a dose of 20 U to 60 U, said botulinum toxin component
selected from the group consisting of a botulinum toxin complex, a
reduced botulinum toxin complex, or a botulinum toxin, a positively
charged carrier component comprising a positively charged
polylysine backbone having covalently attached thereto one or more
positively charged efficiency groups having an amino acid sequence
of (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; and a
pharmaceutically acceptable diluent for injection; wherein the
positively charged carrier is non-covalently associated with the
botulinum toxin component; and wherein said dose of the composition
provides a single treatment having at least about a six month to
about a 10 month duration of effect in reducing the wrinkles,
lines, or furrows in the individual, thereby extending treatment
interval duration for the individual.
5. The method according to claim 1 or claim 2, or the
pharmaceutical composition for use according to claim 3 or claim 4,
wherein the composition reduces glabellar lines in the face of the
individual.
6. The method or pharmaceutical composition for use according to
claim 5, wherein the composition comprises botulinum toxin of
serotype A.
7. The method or pharmaceutical composition for use according to
claim 6, wherein the composition comprises botulinum toxin of
serotype A having a molecular weight of 150 kDa.
8. The method or pharmaceutical composition for use according to
any one of claims 1 to 7, wherein the positively charged polylysine
backbone has covalently attached thereto one or more positively
charged efficiency groups having the amino acid sequence
(gly).sub.p-RGRDDRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20.
9. The method or pharmaceutical composition for use according to
any one of claims 1 to 7, wherein the positively charged polylysine
backbone has covalently attached thereto one or more positively
charged efficiency groups having the amino acid sequence
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20.
10. The method or pharmaceutical composition for use according to
any one of claims 1 to 7, wherein the positively charged polylysine
backbone has covalently attached thereto one or more positively
charged efficiency groups having the amino acid sequence
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20.
11. The method or pharmaceutical composition for use according to
any one of claims 1 to 10, wherein (i) the subscripts p and q are
each independently an integer of from 0 to 8; or (ii) are each
independently an integer of from 2 to 5.
12. The method or pharmaceutical composition for use according to
any one of claims 1 to 11, wherein the one or more positively
charged efficiency groups are attached to either end, or both ends,
of the positively charged polylysine backbone of the positively
charged carrier.
13. The method or pharmaceutical composition for use according to
claim 12, wherein the positively charged carrier has the amino acid
sequence RKKRRQRRRG-(K).sub.15-GRKKRRQRRR.
14. The method or pharmaceutical composition for use according to
any one of claims 1 to 13, wherein the composition does not locally
diffuse from the site of injection following injection.
15. The method or pharmaceutical composition for use according to
any one of claims 1 to 14, wherein the botulinum toxin is
administered to the individual in an amount of 20 U.
16. The method or pharmaceutical composition for use according to
any one of claims 1 to 14, wherein the botulinum toxin is
administered to the individual in an amount of 40 U.
17. The method or pharmaceutical composition for use according to
any one of claims 1 to 14, wherein the botulinum toxin is
administered to the individual in an amount of 60 U.
18. The method according to claim 1 or the or pharmaceutical
composition for use according to claim 3, wherein the therapeutic
effect is reduction of a symptom associated with a disorder
selected from the group consisting of hemifacial spasm, adult onset
spasmodic torticollis, anal fissure, blepharospasm, cerebral palsy,
headaches, strabismus, temporomandibular joint disorder, neurologic
pain, overactive bladder, rhinitis, sinusitis, acne, dystonia,
dystonic contractions, hyperhidrosis, and hypersecretion of a gland
controlled by the cholinergic nervous system.
19. The method according to claim 1, or pharmaceutical composition
for use according to claim 3, wherein the therapeutic or cosmetic
effect is treatment or reduction of wrinkles, lines, or furrows in
the individual.
20. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 3 months.
21. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 4 months.
22. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 5 months.
23. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 6 months.
24. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 7 months.
25. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 8 months.
26. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises greater than 9 months.
27. The method or pharmaceutical composition for use according to
any one of claims 1 to 19, wherein the duration of treatment effect
comprises at least 6 months through 10 months.
28. A sterile injectable composition comprising: a botulinum toxin
component selected from the group consisting of a botulinum toxin,
a botulinum toxin complex, or a reduced botulinum toxin complex, in
a dosage amount selected from 20 U, 40 U, or 60 U; and a positively
charged carrier component comprising a positively charged
polylysine backbone having covalently attached thereto one or more
positively charged efficiency groups having an amino acid sequence
of (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; and a
pharmaceutically acceptable diluent for injection; wherein the
positively charged carrier is non-covalently associated with the
botulinum toxin component; and wherein the composition provides a
cosmetic or therapeutic effect which endures for at least 20 to 24
weeks following a single treatment of an individual with the
injectable composition.
29. The composition according to claim 28, wherein the positively
charged carrier has the amino acid sequence
RKKRRQRRRG-(K).sub.15-GRKKRRQRRR.
30. The composition according to claim 28 or claim 29, wherein the
composition comprises botulinum toxin of serotype A.
31. The composition according to claim 30, wherein the composition
comprises botulinum toxin of serotype A having a molecular weight
of 150 kDa.
32. The composition according to any one of claims 28 to 31,
wherein the botulinum toxin is administered to the individual in a
dosage amount of 20 U.
33. The composition according to any one of claims 28 to 31,
wherein the botulinum toxin is administered to the individual in a
dosage amount of 40 U.
34. The composition according to any one of claims 28 to 31,
wherein the botulinum toxin is administered to the individual in a
dosage amount of 60 U.
35. A method of treating an individual in need of treatment with
injectable botulinum toxin, wherein the method of treatment
comprises a treatment course having multiple treatment intervals
with prolonged duration of effect and duration time between each
treatment interval, the treatment course comprising: administering
by injection an initial treatment dose of a sterile injectable
composition into an area of the individual in need of treatment to
achieve a therapeutic or a cosmetic effect following the initial
treatment with the composition; wherein the composition comprises a
pharmaceutically acceptable diluent suitable for injection; a
botulinum toxin component selected from the group consisting of a
botulinum toxin, a botulinum toxin complex, or a reduced botulinum
toxin complex; and a positively charged carrier component
comprising a positively charged polylysine backbone having
covalently attached thereto one or more positively charged
efficiency groups having an amino acid sequence of
(gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20; and wherein the
botulinum toxin component is administered to the individual in a
treatment dose of 20 U to 60 U; wherein the positively charged
carrier is non-covalently associated with the botulinum toxin
component; wherein the initial treatment dose of the composition
administered by injection to the individual provides a therapeutic
or cosmetic duration of effect lasting through at least about 10
months; and administering subsequent treatment doses of the
composition by injection to the individual at treatment intervals
comprising a duration of greater than or equal to 3 months to at
least about 10 months following the initial treatment dose and
between each subsequent treatment dose.
36. The method according to claim 35, wherein the therapeutic or
cosmetic effect is treatment or reduction of wrinkles, lines, or
furrows.
37. The method according to claim 36, wherein the therapeutic or
cosmetic effect is reduction of glabellar lines in the face of the
individual.
38. The method according to any one of claim 35 to claim 37,
wherein the composition comprises botulinum toxin of serotype
A.
39. The method according to claim 38, wherein the composition
comprises botulinum toxin of serotype A having a molecular weight
of 150 kDa.
40. The method according to any one of claims 35 to 39, wherein the
positively charged polylysine backbone has covalently attached
thereto one or more positively charged efficiency groups having the
amino acid sequence (gly).sub.p-RGRDDRRQRRR-(gly).sub.q, wherein
the subscripts p and q are each independently an integer of from 0
to 20.
41. The method according to any one of claims 35 to 39, wherein the
positively charged polylysine backbone has covalently attached
thereto one or more positively charged efficiency groups having the
amino acid sequence (gly).sub.p-YGRKKRRQRRR-(gly).sub.q, wherein
the subscripts p and q are each independently an integer of from 0
to 20.
42. The method according to any one of claims 35 to 39, wherein the
positively charged polylysine backbone has covalently attached
thereto one or more positively charged efficiency groups having the
amino acid sequence (gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the
subscripts p and q are each independently an integer of from 0 to
20.
43. The method according to any one of claims 35 to 42, wherein (i)
the subscripts p and q are each independently an integer of from 0
to 8; or (ii) are each independently an integer of from 2 to 5.
44. The method according to any one of claims 35 to 43, wherein the
one or more positively charged efficiency groups are attached to
either end, or both ends, of the positively charged polylysine
backbone of the positively charged carrier.
45. The method according to any one of claims 35 to 39, wherein the
positively charged carrier has the amino acid sequence
RKKRRQRRRG-(K).sub.15-GRKKRRQRRR.
46. The method according to any one of claims 35 to 45, wherein the
composition does not locally diffuse from the site of injection
following injection.
47. The method according to any one of claims 35 to 46, wherein the
botulinum toxin is administered to the individual in an amount of
20 U.
48. The method according to any one of claims 35 to 46, wherein the
botulinum toxin is administered to the individual in an amount of
40 U.
49. The method according to any one of claims 35 to 46, wherein the
botulinum toxin is administered to the individual in an amount of
60 U.
50. The method according to claim 35, wherein the therapeutic
effect is reduction of a symptom associated with a disorder
selected from the group consisting of hemifacial spasm, adult onset
spasmodic torticollis, anal fissure, blepharospasm, cerebral palsy,
headaches, strabismus, temporomandibular joint disorder, neurologic
pain, overactive bladder, rhinitis, sinusitis, acne, dystonia,
dystonic contractions, hyperhidrosis, and hypersecretion of a gland
controlled by the cholinergic nervous system.
51. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 3
months.
52. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 4
months.
53. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 5
months.
54. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 6
months.
55. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 7
months.
56. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 8
months.
57. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises greater than 9
months.
58. The method according to any one of claims 35 to 50, wherein the
duration of the treatment interval comprises at least 6 months
through 10 months.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel injectable compositions
comprising botulinum toxin that may be administered to a subject
for various therapeutic, aesthetic and/or cosmetic purposes. The
injectable compositions and methods in which these compositions are
used provide advantageous treatments which result in high responder
rates and long duration of effect, for example, a duration of
effect for over 20 to 24 weeks and longer.
BACKGROUND OF THE INVENTION
[0002] Skin protects the body's organs from external environmental
threats and acts as a thermostat to maintain body temperature. It
consists of several different layers, each with specialized
functions. The major layers include the epidermis, the dermis and
the hypodermis. The epidermis is a stratifying layer of epithelial
cells that overlies the dermis, which consists of connective
tissue. Both the epidermis and the dermis are further supported by
the hypodermis, an internal layer of adipose tissue.
[0003] The epidermis, the topmost layer of skin, is only 0.1 to 1.5
millimeters thick (Inlander, Skin, New York, N.Y.: People's Medical
Society, 1-7 (1998)). It consists of keratinocytes and is divided
into several layers based on their state of differentiation. The
epidermis can be further classified into the stratum corneum and
the viable epidermis, which consists of the granular melphigian and
basal cells. The stratum corneum is hygroscopic and requires at
least 10% moisture by weight to maintain its flexibility and
softness. The hygroscopicity is attributable in part to the
water-holding capacity of keratin. When the horny layer loses its
softness and flexibility it becomes rough and brittle, resulting in
dry skin.
[0004] The dermis, which lies just beneath the epidermis, is 1.5 to
4 millimeters thick. It is the thickest of the three layers of the
skin. Most of the skin's structures, including sweat and oil glands
(which secrete substances through openings in the skin called
pores, or comedos), hair follicles, nerve endings, and blood and
lymph vessels are found in the dermis (Inlander, Skin, New York,
N.Y.: People's Medical Society, 1-7 (1998)). However, the main
components of the dermis are collagen and elastin.
[0005] The hypodermis is the deepest layer of the skin. It acts
both as an insulator for body heat conservation and as a shock
absorber for organ protection (Inlander, Skin, New York, N.Y.:
People's Medical Society, 1-7 (1998)). In addition, the hypodermis
also stores fat for energy reserves. The pH of skin is normally
between 5 and 6. This acidity is due to the presence of amphoteric
amino acids, lactic acid, and fatty acids from the secretions of
the sebaceous glands. The term "acid mantle" refers to the presence
of the water-soluble substances on most regions of the skin. The
buffering capacity of the skin is due in part to these secretions
stored in the skin's horny layer.
[0006] Wrinkles, one of the telltale signs of aging, can be caused
by biochemical, histological, and physiologic changes that
accumulate from environmental damage to the skin. (Benedetto,
International Journal of Dermatology, 38:641-655 (1999)). In
addition, there are other secondary factors that can cause
characteristic folds, furrows, and creases of facial wrinkles
(Stegman et al., The Skin of the Aging Face Cosmetic Dermatological
Surgery, 2.sup.nd ed., St. Louis, Mo.: Mosby Year Book: 5-15
(1990)). These secondary factors include the constant pull of
gravity, frequent and constant positional pressure on the skin
(e.g., during sleep), and repeated facial movements caused by the
contraction of facial muscles (Stegman et al., The Skin of the
Aging Face Cosmetic Dermatological Surgery, 2.sup.nd ed., St.
Louis, Mo.: Mosby Year Book: 5-15 (1990)).
[0007] Different techniques have been utilized in order to
potentially mollify some of the signs of aging. These techniques
range from facial moisturizers containing alpha hydroxy acids and
retinol to surgical procedures and injections of neurotoxins. For
example, in 1986, Jean and Alastair Carruthers, a husband and wife
team consisting of an ocuplastic surgeon and a dermatologist,
developed a method of using the type A form of botulinum toxin for
treatment of movement-associated wrinkles in the glabella area
(Schantz and Scott, In Lewis G. E. (Ed) Biomedical Aspects of
Botulinum, New York: Academic Press, 143-150 (1981)). The
Carruthers' use of the type A form of botulinum toxin for the
treatment of wrinkles led to the seminal publication of this
approach in 1992 (Schantz and Scott, In Lewis G. E. (Ed) Biomedical
Aspects of Botulinum, New York: Academic Press, 143-150 (1981)). By
1994, the same team reported experiences with other
movement-associated wrinkles on the face (Scott, Ophthalmol,
87:1044-1049 (1980)). This in turn led to the birth of the era of
cosmetic treatment using the type A form of botulinum toxin.
[0008] The type A form of botulinum toxin is reported to be the
most lethal natural biological agent known to man. Spores of C.
botulinum are found in soil and can grow in improperly sterilized
and sealed food containers. Botulism, which may be fatal, may be
caused by the ingestion of the bacteria. Botulinum toxin acts to
produce paralysis of muscles by preventing synaptic transmission by
inhibiting the release of acetylcholine across the neuromuscular
junction, and is thought to act in other ways as well. Its action
essentially blocks signals that normally would cause muscle spasms
or contractions, resulting in paralysis. During the last decade,
botulinum toxin's muscle paralyzing activity has been harnessed to
achieve a variety of therapeutic effects. Controlled administration
of botulinum toxin has been used to provide muscle paralysis to
treat a variety of medical conditions, for example, neuromuscular
disorders characterized by hyperactive skeletal muscles. Conditions
that have been treated with botulinum toxin include hemifacial
spasm, adult onset spasmodic torticollis, anal fissure,
blepharospasm, cerebral palsy, cervical dystonia, migraine
headaches, strabismus, temporomandibular joint disorder, and
various types of muscle cramping and spasms. More recently, the
muscle-paralyzing effects of botulinum toxin have been applied to
therapeutic and cosmetic facial applications such as treatment of
wrinkles, frown lines, and other results of spasms or contractions
of facial muscles.
[0009] In addition to the type A form of botulinum toxin, there are
seven other serologically distinct forms of botulinum toxin that
are also produced by the gram-positive bacteria Clostridium
botulinum. Of these eight serologically distinct types of botulinum
toxin, the seven that can cause paralysis have been designated
botulinum toxin serotypes A, B, C, D, E, F and G. Each of these is
distinguished by neutralization with type-specific antibodies. The
molecular weight of each of the botulinum toxin proteins is about
150 kD. Due to the molecule size and molecular structure of
botulinum toxin, it cannot cross stratum corneum and the multiple
layers of the underlying skin architecture. The different serotypes
of botulinum toxin vary in the effect and in the severity and
duration of the paralysis they evoke in different animal species.
For example, in rats, it has been determined that botulinum toxin
type A is 500 times more potent than botulinum toxin type B, as
measured by the rate of paralysis. Additionally, botulinum toxin
type B has been determined to be non-toxic in primates at a dose of
480 U/kg, about 12 times the primate LD.sub.50 for type A.
[0010] As released by Clostridium botulinum bacteria, botulinum
toxin is a component of a toxin complex containing the
approximately 150 kD botulinum toxin protein molecule along with
associated non-toxin proteins. These endogenous non-toxin proteins
are believed to include a family of hemagglutinin proteins, as well
as non-hemagglutinin protein. The non-toxin proteins have been
reported to stabilize the botulinum toxin molecule in the toxin
complex and protect it against denaturation by digestive acids when
toxin complex is ingested. Thus, the non-toxin proteins of the
toxin complex protect the activity of the botulinum toxin and
thereby enhance systemic penetration when the toxin complex is
administered via the gastrointestinal tract. Additionally, it is
believed that some of the non-toxin proteins specifically stabilize
the botulinum toxin molecule in blood.
[0011] The presence of non-toxin proteins in the toxin complexes
typically causes the toxin complexes to have molecular weights that
are greater than that of the bare botulinum toxin molecule, which
is about 150 kD, as previously stated. For example, Clostridium
botulinum bacteria can produce botulinum type A toxin complexes
that have molecular weights of about 900 kD, 500 kD or 300 kD.
Botulinum toxin types B and C are produced as complexes having a
molecular weight of about 700 kD or about 500 kD. Botulinum toxin
type D is produced as complexes having molecular weights of about
300 kD or 500 kD. Botulinum toxin types E and F are only produced
as complexes having a molecular weight of about 300 kD.
[0012] To provide additional stability to botulinum toxin, the
toxin complexes are conventionally stabilized by combining the
complexes with albumin during manufacturing. For example,
BOTOX.RTM. (Allergan, Inc., Irvine, Calif.) is a botulinum
toxin-containing formulation that contains 100 U of type A
botulinum toxin with accessory proteins, 0.5 milligrams of human
albumin, and 0.9 milligrams of sodium chloride. The albumin serves
to bind and to stabilize toxin complexes in disparate environments,
including those associated with manufacturing, transportation,
storage, and administration.
[0013] Typically, the botulinum toxin is administered to patients
by carefully controlled injections of compositions containing
botulinum toxin complex and albumin. However, there are several
problems associated with this approach. Not only are the injections
painful, but typically large subdermal wells of toxin are locally
generated around the injection sites, in order to achieve the
desired therapeutic or cosmetic effect. The botulinum toxin may
migrate from these subdermal wells to cause unwanted paralysis in
surrounding areas of the body. This problem is exacerbated when the
area to be treated is large and many injections of toxin are
required to treat the area. Moreover, because the injected toxin
complexes contain non-toxin proteins and albumin that stabilize the
botulinum toxin and increase the molecular weight of the toxin
complex, the toxin complexes have a long half-life in the body and
may cause an undesirable antigenic response in the patient. For
example, some patients will, over time, develop an allergy to the
albumin used as a stabilizer in current commercial formulations.
Also, the toxin complexes may induce the immune system of the
patient to form neutralizing antibodies, so that larger amounts of
toxin are required in subsequent administrations to achieve the
same effect. When this happens, subsequent injections must be
carefully placed so that they do not release a large amount of
toxin into the bloodstream of the patient, which could lead to
fatal systemic poisoning, especially since the non-toxin proteins
and albumin stabilize the botulinum toxin in blood.
[0014] In view of the drawbacks associated with current botulinum
toxin formulations, it would be highly desirable to have an
injectable botulinum toxin formulation that is efficacious and
stable, but exhibits reduced antigenicity and a lower tendency to
diffuse locally after injection. It would also be desirable to use
such a botulinum toxin formulation for various therapeutic,
aesthetic and/or cosmetic purposes.
SUMMARY OF THE INVENTION
[0015] In one of its aspects, this invention provides injectable
compositions comprising botulinum toxin non-covalently associated
with a positively charged carrier molecule. In preferred
embodiments, the compositions of the invention possess one or more
advantages over conventional commercial botulinum toxin
formulations, such as BOTOX.RTM. or MYOBLOC.RTM.. For instance, in
certain embodiments, the compositions may exhibit one or more
advantages over conventional injectable botulinum formulations,
including reduced antigenicity, a reduced tendency to undergo
diffusion into surrounding tissue following injection, increased
duration of clinical efficacy or enhanced potency relative to
conventional botulinum toxin formulations, faster onset of clinical
efficacy, and/or improved stability.
[0016] Another aspect of this invention is the recognition that
certain non-native molecules (i.e., molecules not found in
botulinum toxin complexes obtained from Clostridium botulinum
bacteria) can be added to botulinum toxin, botulinum toxin
complexes, and in particular reduced botulinum toxin complexes (as
defined herein), to improve toxin diffusion through tissues. The
non-native molecules associate non-covalently with the toxin and
act as penetration enhancers that improve the ability of the toxin
to reach target structures after injection. Furthermore, the
non-native molecules may increase the stability of the toxin prior
to and after injection. By way of example, the penetration
enhancers may be positively charged carriers, such as cationic
peptides, which have no inherent botulinum-toxin-like activity and
which also contain one or more protein transduction domains as
described herein.
[0017] Another aspect of this invention is to provide a composition
comprising botulinum toxin, a botulinum toxin complex (or a reduced
protein botulinum toxin complex including just the 150 kD
neurotoxin itself, or the neurotoxin with some, but not all, of the
native complex proteins) and a positively charged carrier.
[0018] In another of its aspects, the invention further relates to
a method for producing a biologic effect by injecting an effective
amount, preferably a therapeutically effective amount, of the
compositions of this invention to a subject or patient in need of
such treatment. The biologic effect may include, for example,
muscle paralysis, reduction of hypersecretion or sweating,
treatment of neurologic pain or migraine headache, management of
rhinitis or sinusitis, treatment of hyperactive bladder, reduction
of muscle spasms, prevention or reduction of acne, reduction or
enhancement of an immune response, reduction of wrinkles, fine
lines, or glabellar lines, also known as frown lines, particularly
in the face, or prevention or treatment of various other
disorders.
[0019] In another aspect, the invention provides effective doses
and amounts of the compositions of this invention that afford a
long-lasting, sustained efficacy e.g., a response rate of long
duration, following administration by injection to a subject or
patient in need of treatment. Such doses and amounts are preferably
therapeutically or cosmetically effective doses and amounts that
produce or result in a desired therapeutic or cosmetic effect in a
subject to whom the doses and amounts are administered. In an
embodiment, the effect of reducing wrinkles and lines of the face,
particularly, glabellar lines, provided by a dose of a composition
of the invention administered by injection as a single treatment to
the subject or patient in need, lasted several weeks to several
months compared with conventional treatment, such as BOTOX.RTM.
Cosmetic injection. In particular embodiments, and as described in
the Examples herein, a single treatment of a subject or patient
with a composition of the invention comprising a botulinum toxin,
such as botulinum toxin A, and a positively charged carrier, as
described herein, in therapeutically effective amounts of 20 U to
60 U, afforded a response rate of wrinkle and facial line reduction
for at least 20 weeks, at least 24 weeks, or about 6 to 10 months,
or even longer, compared with conventional BOTOX.RTM. injection.
Moreover, the compositions of the invention provide an attribute of
reduced diffusion or spread from the injection site following
injection, thereby localizing the toxin and its effect where
desired and decreasing nonspecific or unwanted effects of the toxin
at sites or locations distant from the site of injection for
treatment.
[0020] The duration of effect provided by compositions of the
invention, e.g., RT002 of Example 5, as well as by the described
treatment methods and uses, affords significant advantages compared
to the art. By way of example, subjects undergoing treatment, such
as aesthetic treatment, with compositions containing botulinum
toxin consider that duration of effect following treatment is of
high importance to them. Such a long, sustained duration of effect,
which is achieved by even a single treatment with an effective dose
of a product of the invention, for example, RT002, permits fewer
injections per treatment course for a subject, which is extremely
important for the subject. A prolonged duration effect from a
single treatment with a product which has clear efficacy and
safety, as provided by the inventive compositions and methods
described herein, offer less discomfort, less cost and more
convenience to subjects undergoing a course of treatment.
Furthermore, a product that affords significant and sustained
effects, which are maintained for at least a 20 or 24 week period,
or for at least a 6-month period, or for greater than a 6-month
period, following the single injectable treatment of the product to
a subject, provides a solution to an unmet need in the art for both
practitioners and patients alike. Thus, the compositions and
methods of the invention provide a solution to the problem of too
frequent treatments and improve patients' overall well-being. Such
prolonged duration of action provides for fewer treatments over an
entire treatment course.
[0021] In another aspect, the invention provides a method of
administering botulinum toxin to achieve an extended duration
therapeutic or cosmetic effect in an individual, in which the
method comprises administering by injection a dose of a sterile
injectable composition into an area of the individual in need of
treatment to achieve the therapeutic or cosmetic effect following a
first treatment with the composition; wherein the composition
comprises a botulinum toxin, a botulinum toxin complex, or a
reduced botulinum toxin complex component and a positively charged
carrier component comprising a positively charged polylysine
backbone having covalently attached thereto one or more positively
charged efficiency groups having an amino acid sequence of
(gly)p-RGRDDRRQRRR-(gly)q, (gly)p-YGRKKRRQRRR-(gly)q or
(gly)p-RKKRRQRRR-(gly)q, wherein the subscripts p and q are each
independently an integer of from 0 to 20; wherein the botulinum
toxin, botulinum toxin complex, or reduced botulinum toxin complex
component is administered to the individual in a treatment dose of
20 U to 60 U; wherein the positively charged carrier is
non-covalently associated with the botulinum toxin, botulinum toxin
complex, or reduced botulinum toxin complex component; and a
pharmaceutically acceptable diluent suitable for injection; and
wherein the first treatment dose of the composition administered by
injection to the individual achieves the extended duration
therapeutic or cosmetic effect having at least about a 6 month to
about a 10 month duration of effect, optionally, before a second or
subsequent treatment dose is administered.
[0022] In another aspect, the invention provides a method of
reducing wrinkles, lines, or furrows in an individual in need
thereof, the method comprising administering to the individual as a
single dose injection a composition comprising: a botulinum toxin,
a botulinum toxin complex, or a reduced botulinum toxin complex and
a positively charged carrier comprising a positively charged
polylysine backbone having covalently attached thereto one or more
positively charged efficiency groups having an amino acid sequence
of (gly)p-RGRDDRRQRRR-(gly)q, (gly)p-YGRKKRRQRRR-(gly)q or
(gly)p-RKKRRQRRR-(gly)q, wherein the subscripts p and q are each
independently an integer of from 0 to 20; and a pharmaceutically
acceptable diluent for injection; wherein the botulinum toxin is
administered to the individual in an amount of 20 U to 60 U;
wherein the positively charged carrier is non-covalently associated
with the botulinum toxin, botulinum toxin complex, or reduced
botulinum toxin complex component; and wherein the single dose
injection of the composition provides a single treatment having at
least about a six month to about a 10 month duration of effect in
reducing the wrinkles, lines, or furrows in the individual, thereby
extending treatment interval duration for the individual. In an
embodiment, the composition reduces, or reduces the severity of,
glabellar lines in the face of the individual.
[0023] In another aspect, the invention provides a sterile
injectable composition comprising a botulinum toxin, a botulinum
toxin complex, or a reduced botulinum toxin complex in a dosage
amount selected from 20 U, 40 U, or 60 U; and a positively charged
carrier comprising a positively charged polylysine backbone having
covalently attached thereto one or more positively charged
efficiency groups having an amino acid sequence of
(gly)p-RGRDDRRQRRR-(gly)q, (gly)p-YGRKKRRQRRR-(gly)q or
(gly)p-RKKRRQRRR-(gly)q, wherein the subscripts p and q are each
independently an integer of from 0 to 20; and a pharmaceutically
acceptable diluent for injection; wherein the positively charged
carrier is non-covalently associated with the botulinum toxin,
botulinum toxin complex, or reduced botulinum toxin complex
component; and wherein the composition provides a cosmetic or
therapeutic effect which endures for at least 20 to 24 weeks, or
for at least 6 months, or greater than 6 months, e.g., about 6
months to about 10 months, following a treatment of an individual
with an effective dose of the injectable composition.
[0024] In some embodiments of these above methods and composition,
the composition comprises botulinum toxin of serotype A, preferably
a serotype A botulinum toxin having a molecular weight of 150 kDa.
In an embodiment, the positively charged carrier has the amino acid
sequence RKKRRQRRRG-(K).sub.15-GRKKRRQRRR. In an embodiment, the
botulinum toxin is present in the composition in a dosage amount of
20 U. In an embodiment, the botulinum toxin is present in the
composition in a dosage amount of 40 U. In an embodiment, the
botulinum toxin is present in the composition in a dosage amount of
60 U. In an embodiment, the composition reduces the severity of
glabellar lines in an individual who has undergone a single
treatment by injection of the composition. In certain embodiments,
the duration of the treatment effect comprises greater than 6
months; greater than 7 months; greater than 8 months; greater than
9 months; or at least 6 months through 10 months.
[0025] In another of its aspects, the invention provides a method
of treating an individual who is in need of treatment with
injectable botulinum toxin, in which the method of treatment
comprises a treatment course having multiple treatment intervals
with prolonged duration of effect and duration time between each
treatment interval, the treatment course comprising: administering
by injection an initial treatment dose of a sterile injectable
composition into an area of the individual in need of treatment to
achieve a therapeutic or a cosmetic effect following the initial
treatment with the composition; wherein the composition comprises a
botulinum toxin, a botulinum toxin complex, or a reduced botulinum
toxin complex component and a positively charged carrier component
comprising a positively charged polylysine backbone having
covalently attached thereto one or more positively charged
efficiency groups having an amino acid sequence of
(gly)p-RGRDDRRQRRR-(gly)q, (gly)p-YGRKKRRQRRR-(gly)q or
(gly)p-RKKRRQRRR-(gly)q, wherein the subscripts p and q are each
independently an integer of from 0 to 20; and a pharmaceutically
acceptable diluent suitable for injection; wherein the botulinum
toxin, botulinum toxin complex, or reduced botulinum toxin complex
component is administered to the individual in a treatment dose of
20 U to 60 U; wherein the positively charged carrier is
non-covalently associated with the botulinum toxin, botulinum toxin
complex, or reduced botulinum toxin complex component; wherein the
initial treatment dose of the composition administered by injection
to the individual provides a therapeutic or cosmetic duration of
effect lasting through at least about 10 months; and administering
subsequent treatment doses of the composition by injection to the
individual at treatment intervals comprising a duration of greater
than or equal to 3 months to at least about 10 months following the
initial treatment dose and between each subsequent treatment
dose.
[0026] In embodiments of the above-described treatment method, the
therapeutic or cosmetic effect is treatment or reduction of
wrinkles, lines, or furrows. In an embodiment, the therapeutic or
cosmetic effect is reduction of glabellar lines in the face of the
individual. In an embodiment, the composition comprises botulinum
toxin of serotype A, preferably, botulinum toxin of serotype A
having a molecular weight of 150 kDa. In an embodiment, the
positively charged carrier is a positively charged peptide having
the amino acid sequence RKKRRQRRRG-(K)15-GRKKRRQRRR. In an
embodiment, the composition does not locally diffuse from the site
of injection following injection. In specific embodiments, the
botulinum toxin is administered to the individual in an amount of
20 U, or in an amount of 40 U, or in an amount of 60 U. In an
embodiment, the therapeutic effect is reduction of a symptom
associated with a disorder selected from the group consisting of
hemifacial spasm, adult onset spasmodic torticollis, anal fissure,
blepharospasm, cerebral palsy, headaches, strabismus,
temporomandibular joint disorder, neurologic pain, overactive
bladder, rhinitis, sinusitis, acne, dystonia, dystonic
contractions, hyperhidrosis, and hypersecretion of a gland
controlled by the cholinergic nervous system. In certain
embodiments, the duration of the treatment interval comprises
greater than 3 months; greater than 4 months; greater than 5
months; greater than 6 months; greater than 7 months; greater than
8 months; greater than 9 months; or at least 6 months through 10
months.
[0027] This invention also provides kits for preparing formulations
or compositions containing a botulinum toxin, a botulinum toxin
complex, or a reduced protein botulinum toxin complex and
positively charged carrier, or a premix that may in turn be used to
produce such a formulation or composition. Also provided are kits
that contain means for sequentially administering a botulinum toxin
complex (or a reduced botulinum toxin complex including just the
150 KD neurotoxin itself or the neurotoxin with some native complex
proteins) and a positively charged carrier.
DESCRIPTION OF THE FIGURES
[0028] FIG. 1 presents a bar graph showing the required time to
return to the baseline digit abduction score (DAS) value (0.4)
following repeated administration of either RT003 or
BOTOX.RTM..
[0029] FIGS. 2A and 2B: FIG. 2A shows the hind leg of a mouse
injected with a dark dye to indicate the portion of a mouse's
gastrocnemius muscle that is affected by lateral-to-midline
injection. FIG. 2B shows the hind leg of a mouse injected with a
dark dye to indicate the portion of a mouse's gastrocnemius muscle
that is affected by midline injection.
[0030] FIG. 3 presents digital abduction scores (DAS) measured as a
function of time following injection of RT003, RTT150, or
BOTOX.RTM. into either the lateral-to-midline or midline portion of
the gastrocnemius muscle of a mouse.
[0031] FIGS. 4A and 4B present Kaplan-Meier Curves showing the
duration of response in various treatment groups from the clinical
study described in Example 5 herein. The Kaplan-Meier Curves
represent results from primary efficacy analyses of the clinical
study and demonstrate duration of response for at least a 1-point
improvement from baseline for Investigator Global Assessment-Facial
Wrinkle Severity (IGA-FWS) Assessment in the indicated treatment
groups. FIG. 4A presents Kaplan-Meier Curves for the Placebo
treatment group, the VISTABEL.RTM./BOTOX.RTM. 20 U treatment group
and the RT002 40 U treatment group. FIG. 4B presents Kaplan-Meier
Curves for the Placebo treatment group, the
VISTABEL.RTM./BOTOX.RTM. 20 U treatment group, the RT002 20 U
treatment group, the RT002 40 U treatment group and the RT002 60 U
treatment group.
DETAILED DESCRIPTION OF THE INVENTION
[0032] This invention relates to novel injectable compositions
comprising botulinum toxin, a botulinum toxin complex, or a reduced
botulinum toxin complex. In preferred embodiments, the compositions
stabilize the toxin or enable the transport or delivery of toxin
through tissues after injection such that the toxin has reduced
antigenicity, a better safety profile, enhanced potency, faster
onset of clinical efficacy and/or longer duration of clinical
efficacy compared to conventional commercial botulinum toxin
complexes that are bound to exogenous albumin (e.g., BOTOX.RTM. or
MYOBLOC.RTM.). The compositions of the invention may be used as
injectable applications for providing a botulinum toxin to a
subject, for various therapeutic, aesthetic and/or cosmetic
purposes, as described herein. The compositions of the invention
also have an improved safety profile over other compositions and
methods of delivery of botulinum toxin. In addition, these
compositions can afford beneficial reductions in immune responses
to the botulinum toxin. In embodiments, the injectable compositions
of the invention provide long lasting efficacy, e.g., an effect
lasting at least 20 weeks, at least 24 weeks, at least 6 months, or
greater than 6 months, for example, up to about 10 months, in
subjects to whom such compositions, particularly those comprising
botulinum toxin in amounts of 20 U or more, are administered by
injection for the treatment of wrinkles and facial lines, such as
glabellar lines.
[0033] The term "botulinum toxin" as used herein may refer to any
of the known types of botulinum toxin (e.g., 150 kD botulinum toxin
protein molecules associated with the different serotypes of C.
botulinum), whether produced by the bacterium or by recombinant
techniques, as well as any such types that may be subsequently
discovered including newly discovered serotypes, and engineered
variants or fusion proteins. As mentioned above, currently seven
immunologically distinct botulinum neurotoxins have been
characterized, namely botulinum neurotoxin serotypes A, B, C, D, E,
F and G, each of which is distinguished by neutralization with
type-specific antibodies. The botulinum toxin serotypes are
commercially available, for example, from Sigma-Aldrich (St. Louis,
Mo.) and from Metabiologics, Inc. (Madison, Wis.), as well as from
other sources. 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. At least two types of
botulinum toxin, types A and B, are available commercially in
formulations for treatment of certain conditions. Type A, for
example, is contained in preparations of Allergan having the
trademark BOTOX.RTM. and of Ipsen having the trademark
DYSPORT.RTM., and type B is contained in preparations of Elan
having the trademark MYOBLOC.RTM..
[0034] The term "botulinum toxin" used in the compositions of this
invention can alternatively refer to a botulinum toxin derivative,
that is, a compound that has botulinum toxin activity but contains
one or more chemical or functional alterations on any part or on
any amino acid chain relative to naturally occurring or recombinant
native botulinum toxins. For instance, the botulinum toxin may be a
modified neurotoxin that is a neurotoxin which has at least one of
its amino acids deleted, modified or replaced, as compared to a
native form, or the modified neurotoxin can be a recombinantly
produced neurotoxin or a derivative or fragment thereof. For
instance, the botulinum toxin may be one that has been modified in
a way that, for instance, enhances its properties or decreases
undesirable side effects, but that still retains the desired
botulinum toxin activity. Alternatively the botulinum toxin used in
this invention may be a toxin prepared using recombinant or
synthetic chemical techniques, e.g. a recombinant peptide, a fusion
protein, or a hybrid neurotoxin, for example prepared from subunits
or domains of different botulinum toxin serotypes (See, U.S. Pat.
No. 6,444,209, for instance). The botulinum toxin may also be a
portion of the overall molecule that has been shown to possess the
necessary botulinum toxin activity, and in such case may be used
per se or as part of a combination or conjugate molecule, for
instance a fusion protein. Alternatively, the botulinum toxin may
be in the form of a botulinum toxin precursor, which may itself be
non-toxic, for instance a non-toxic zinc protease that becomes
toxic on proteolytic cleavage.
[0035] The term "botulinum toxin complex" or "toxin complex" as
used herein refers to the approximately 150 kD botulinum toxin
protein molecule (belonging to any one of botulinum toxin serotypes
A-G), along with associated endogenous non-toxin proteins (i.e.,
hemagglutinin protein and non-toxin non-hemagglutinin protein
produced by Clostridium botulinum bacteria). Note, however, that
the botulinum toxin complex need not be derived from Clostridium
botulinum bacteria as one unitary toxin complex. For example,
botulinum toxin or modified botulinum toxin may be recombinantly
prepared first and then subsequently combined with the non-toxin
proteins. Recombinant botulinum toxin can also be purchased (e.g.,
from List Biological Laboratories, Campbell, Calif.) and then
combined with non-toxin proteins.
[0036] This invention also contemplates modulation of the stability
of botulinum toxin molecules through the addition of one or more
exogenous stabilizers, the removal of endogenous stabilizers, or a
combination thereof. For example, this invention contemplates the
use of "reduced botulinum toxin complexes", in which the botulinum
toxin complexes have reduced amounts of non-toxin protein compared
to the amounts naturally found in botulinum toxin complexes
produced by Clostridium botulinum bacteria. In one embodiment,
reduced botulinum toxin complexes are prepared using any
conventional protein separation method to extract a fraction of the
hemagglutinin protein or non-toxin non-hemagglutinin protein from
botulinum toxin complexes derived from Clostridium botulinum
bacteria. For example, reduced botulinum toxin complexes may be
produced by dissociating botulinum toxin complexes through exposure
to red blood cells at a pH of 7.3 (e.g., see EP 1514556 A1, hereby
incorporated herein by reference). HPLC, dialysis, columns,
centrifugation, and other methods for extracting proteins from
proteins can be used. Alternatively, when the reduced botulinum
toxin complexes are to be produced by combining synthetically
produced botulinum toxin with non-toxin proteins, one may simply
add less hemagglutinin or non-toxin, non-hemagglutinin protein to
the mixture than what would be present for naturally occurring
botulinum toxin complexes. Any of the non-toxin proteins (e.g.,
hemagglutinin protein or non-toxin non-hemagglutinin protein or
both) in the reduced botulinum toxin complexes according to the
invention may be reduced independently by any amount. In certain
exemplary embodiments, one or more non-toxin proteins are reduced
by at least about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or 100% compared to the amounts normally found in
botulinum toxin complexes. As noted above, Clostridium botulinum
bacteria produce seven different serotypes of toxin and commercial
preparations are manufactured with different relative amounts of
non-toxin proteins (i.e. different amount of toxin complexes). For
example, MYOBLOC.TM. has 5000 U of Botulinum toxin type B per ml
with 0.05% human serum albumin, 0.01 M sodium succinate, and 0.1 M
sodium chloride. DYSPORT.TM. has 500 U of botulinum toxin type
A-hemagglutinin complex with 125 mcg albumin and 2.4 mg lactose. In
certain embodiments, substantially all of the non-toxin protein
(e.g., greater than 95%, 96%, 97%, 98% or 99% of the hemagglutinin
protein and non-toxin non-hemagglutinin protein) that would
normally be found in botulinum toxin complexes derived from
Clostridium botulinum bacteria is removed from the botulinum toxin
complex. Furthermore, although the amount endogenous non-toxin
proteins may be reduced by the same amount in some cases, this
invention also contemplates reducing each of the endogenous
non-toxin proteins by different amounts, as well as reducing at
least one of the endogenous non-toxin proteins, but not the
others.
[0037] As noted above, an exogenous stabilizer (e.g., albumin) is
typically added to stabilize botulinum toxin formulations. For
instance, in the case of BOTOX.RTM., 0.5 mg of human albumin per
100 U of type A botulinum toxin complex to stabilize the complex.
Generally, the amount of exogenous stabilizer that may be added to
stabilize the compositions according to the invention is not
particularly limited. In some embodiments, the amount of added
stabilizer may be less than the amount conventionally added, owing
to the ability of positively charged carriers of the invention to
act as a stabilizer in its own right. For instance, the amount of
added exogenous albumin can be any amount less than the
conventional thousand-fold excess of exogenous albumin and, in
certain exemplary embodiments of the invention, is only about 0.25,
0.20, 0.15, 0.10, 0.01, 0.005, 0.001, 0.0005, 0.00001, 0.000005,
0.000001, or 0.0000001 mg per 100 U of botulinum toxin. In one
embodiment, no exogenous albumin is added as a stabilizer to the
compositions of the invention, thus producing albumin-free
botulinum toxin compositions.
[0038] A preferred composition of the invention is a liquid,
botulinum toxin-containing composition that is stabilized without a
proteinaceous excipient, especially without any animal
protein-derived excipients. Such a liquid composition comprises a
botulinum toxin, preferably botulinum toxin of serotype A, a
positively charged carrier (e.g., peptide) a non-reducing
disaccharide or a non-reducing trisaccharide, a non-ionic
surfactant, and a physiologically compatible buffer for maintaining
the pH between 4.5. and 7.5. The concentration of the non-reducing
sugar in the liquid composition is in the range of 10% through 40%
(w/v) and the concentration of the non-ionic surfactant is in the
range of 0.005% through 0.5% (w/v). The preferred composition
provides a long duration effect after treatment by a single
injection In a preferred embodiment, the botulinum toxin A has a
molecular weight (MW) of 150 kDa. The preferred composition
comprises botulinum toxin, preferably botulinum toxin A, more
preferably, of 150 kDa MW, a positively charged carrier (e.g.,
peptide) as described herein, a non-reducing disaccharide, such as
sucrose, a non-ionic surfactant, such as polysorbate 20,
polysorbate 40, polysorbate 60, polysorbate 80, or a sorbitan
ester, and a physiologically compatible buffer, such as citric
acid, acetic acid, succinic acid, tartaric acid, maleic acid, and
histidine; and has a pH in the range of pH 4.5. to pH 7.5.
[0039] According to the present invention, a positively charged
carrier molecule having protein transduction domains or efficiency
groups, as described herein, has been found suitable as a transport
system for a botulinum toxin, enabling toxin to be injected with
improved penetration to target structures such as muscles and/or
other skin-associated structures. The transport occurs without
covalent modification of the botulinum toxin. Besides enhancing
penetration of botulinum toxin, the positively charged carriers of
the invention may, in certain preferred embodiments, stabilize the
botulinum toxin against degradation. In such embodiments, the
hemagglutinin protein and non-toxin, non-hemagglutinin protein that
are normally present to stabilize the botulinum toxin may be
reduced or omitted entirely. Similarly, the exogenous albumin that
is normally added during manufacturing may be omitted.
[0040] By the use of the terms "positively charged" or "cationic"
in connection with the term "carrier", it is meant that the carrier
has a positive charge under at least some solution-phase
conditions, more preferably, under at least some physiologically
compatible conditions. More specifically, "positively charged" and
"cationic" as used herein, means that the group in question
contains functionalities that are charged under all pH conditions,
for instance, a quaternary amine, or contains a functionality which
can acquire positive charge under certain solution-phase
conditions, such as pH changes in the case of primary amines. More
preferably, "positively charged" or "cationic" as used herein
refers to those groups that have the behavior of associating with
anions over physiologically compatible conditions. Polymers with a
multiplicity of positively-charged moieties need not be
homopolymers, as will be apparent to one skilled in the art. Other
examples of positively charged moieties are well known in the prior
art and can be employed readily, as will be apparent to those
skilled in the art.
[0041] Generally, the positively-charged carrier (also referred to
as a "positively charged backbone") is typically a chain of atoms,
either with groups in the chain carrying a positive charge at
physiological pH, or with groups carrying a positive charge
attached to side chains extending from the backbone. In certain
preferred embodiments, the positively charged backbone is a
cationic peptide. As used herein, the term "peptide" refers to an
amino acid sequence, but carries no connotation with respect to the
number of amino acid residues within the amino acid sequence.
Accordingly, the term "peptide" may also encompass polypeptides and
proteins. In certain preferred embodiments, the positively charged
backbone itself will not have a defined enzymatic or therapeutic
biologic activity. In certain embodiments, the backbone is a linear
hydrocarbon backbone which is, in some embodiments, interrupted by
heteroatoms selected from nitrogen, oxygen, sulfur, silicon and
phosphorus. The majority of backbone chain atoms are usually
carbon. Additionally, the backbone will often be a polymer of
repeating units (e.g., amino acids, poly(ethyleneoxy),
poly(propyleneamine), polyalkyleneimine, and the like) but can be a
heteropolymer. In one group of embodiments, the positively charged
backbone is a polypropyleneamine wherein a number of the amine
nitrogen atoms are present as ammonium groups (tetra-substituted)
carrying a positive charge. In another embodiment, the positively
charged backbone is a nonpeptidyl polymer, which may be a hetero-
or homo-polymer such as a polyalkyleneimine, for example a
polyethyleneimine or polypropyleneimine, having a molecular weight
of from about 10,000 to about 2,500,000, preferably from about
100,000 to about 1,800,000, and most preferably from about 500,000
to about 1,400,000. In another group of embodiments, the backbone
has attached a plurality of side-chain moieties that include
positively charged groups (e.g., ammonium groups, pyridinium
groups, phosphonium groups, sulfonium groups, guanidinium groups,
or amidinium groups). The sidechain moieties in this group of
embodiments can be placed at spacings along the backbone that are
consistent in separations or variable. Additionally, the length of
the sidechains can be similar or dissimilar. For example, in one
group of embodiments, the sidechains can be linear or branched
hydrocarbon chains having from one to twenty carbon atoms and
terminating at the distal end (away from the backbone) in one of
the above-noted positively charged groups. The association between
the positively charged carrier and the botulinum toxin is by
non-covalent interaction, non-limiting examples of which include
ionic interactions, hydrogen bonding, van der Waals forces, or
combinations thereof.
[0042] In one group of embodiments, the positively charged backbone
is a polypeptide having multiple positively charged sidechain
groups (e.g., lysine, arginine, ornithine, homoarginine, and the
like). Preferably, the polypeptide has a molecular weight from
about 100 to about 1,500,000, more preferably from about 500 to
about 1,200,000, most preferably from about 1000 to about
1,000,000. One of skill in the art will appreciate that when amino
acids are used in this portion of the invention, the sidechains can
have either the D- or L-form (R or S configuration) at the center
of attachment. In certain preferred embodiments, the polypeptide
has a molecular weight from about 500 to about 5000, more
preferably from 1000 to about 4000, more preferably from 2000 to
about 3000. In other preferred embodiments, the polypeptide
comprises 10 to 20 amino acids, or 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acids, preferably polylysine.
[0043] Alternatively, the backbone may comprise amino acid analogs
and/or synthetic amino acids. The backbone may also be an analog of
a polypeptide such as a peptoid. See, for example, Kessler, Angew.
Chem. Int. Ed. Engl. 32:543 (1993); Zuckermann et al.
Chemtracts-Macromol. Chem. 4:80 (1992); and Simon et al. Proc.
Nat'l. Acad. Sci. USA 89:9367 (1992)). Briefly, a peptoid is a
polyglycine in which the sidechain is attached to the backbone
nitrogen atoms rather than the .alpha.-carbon atoms. As above, a
portion of the sidechains will typically terminate in a positively
charged group to provide a positively charged backbone component.
Synthesis of peptoids is described in, for example, U.S. Pat. No.
5,877,278, which is hereby incorporated by reference in its
entirety. As the term is used herein, positively charged backbones
that have a peptoid backbone construction are considered
"non-peptide" as they are not composed of amino acids having
naturally occurring sidechains at the alpha-carbon locations.
[0044] A variety of other backbones can be used employing, for
example, steric or electronic mimics of polypeptides wherein the
amide linkages of the peptide are replaced with surrogates such as
ester linkages, thioamides (--CSNH--), reversed thioamide
(--NHCS--), aminomethylene (--NHCH.sub.2--) or the reversed
methyleneamino (--CH.sub.2NH--) groups, keto-methylene
(--COCH.sub.2--) groups, phosphinate (--PO.sub.2RCH.sub.2--),
phosphonamidate and phosphonamidate ester (--PO.sub.2RNH--),
reverse peptide (--NHCO--), trans-alkene (--CR.dbd.CH--),
fluoroalkene (--CF.dbd.CH--), dimethylene (--CH.sub.2CH.sub.2--),
thioether (--CH.sub.2S--), hydroxyethylene (--CH(OH)CH.sub.2--),
methyleneoxy (--CH.sub.2O--), tetrazole (CN.sub.4), sulfonamido
(--SO.sub.2NH--), methylenesulfonamido (--CHRSO.sub.2NH--),
reversed sulfonamide (--NHSO.sub.2--), and backbones with malonate
and/or gem-diamino-alkyl subunits, for example, as reviewed by
Fletcher et al. ((1998) Chem. Rev. 98:763) and detailed by
references cited therein. Many of the foregoing substitutions
result in approximately isosteric polymer backbones relative to
backbones formed from .alpha.-amino acids.
[0045] In each of the backbones provided above, sidechain groups
can be appended that carry a positively charged group. For example,
the sulfonamide-linked backbones (--SO.sub.2NH-- and
--NHSO.sub.2--) can have sidechain groups attached to the nitrogen
atoms. Similarly, the hydroxyethylene (--CH(OH)CH.sub.2--) linkage
can bear a sidechain group attached to the hydroxy substituent. One
of skill in the art can readily adapt the other linkage chemistries
to provide positively charged sidechain groups using standard
synthetic methods.
[0046] In one embodiment, the positively charged backbone is a
polypeptide having protein transduction domains (also referred to
as efficiency groups). As used herein, an efficiency group or
protein transduction domain is any agent that has the effect of
promoting the translocation of the positively charged backbone
through a tissue or cell membrane. Non-limiting examples of protein
transduction domains or efficiency groups include
-(gly).sub.n1-(arg), HIV-TAT or fragments thereof, or the protein
transduction domain (PTD) of Antennapedia, or a fragment thereof,
in which the subscript n1 is an integer of from 0 to 20, more
preferably 0 to 8, still more preferably 2 to 5, and the subscript
n2 is independently an odd integer of from about 5 to about 25,
more preferably about 7 to about 17, most preferably about 7 to
about 13. In some embodiments, the HIV-TAT fragment does not
contain the cysteine-rich region of the HIV-TAT molecule, in order
to minimize the problems associated with disulfide aggregation.
Preferably, the fragments of the HIV-TAT and Antennapedia protein
transduction domains retain the protein transduction activity of
the full protein. Still further preferred are those embodiments in
which the HIV-TAT fragment has the amino acid sequence
(gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q wherein the subscripts p and q
are each independently an integer of from 0 to 20, or wherein p and
q are each independently the integer 1. In another embodiment, the
fragment or efficiency group is attached to the backbone via either
the C-terminus or the N-terminus of the fragment or amino acid
sequence of the efficiency group. In certain preferred embodiments,
p is one and q is zero or p is zero and q is one. Preferred HIV-TAT
fragments are those in which the subscripts p and q are each
independently integers of from 0 to 8, more preferably 0 to 5. In
another preferred embodiment the positively charged side chain or
branching group is the Antennapedia (Antp) protein transduction
domain (PTD), or a fragment thereof that retains activity. These
are known in the art, for instance, from Console et al., J. Biol.
Chem. 278:35109 (2003) and a non-limiting example of an
Antennapedia PTD contemplated by this invention is the PTD having
the amino acid sequence SGRQIKIWFQNRRMKWKKC. In other embodiments,
the positively charged carrier is a positively charged peptide
having the amino acid sequence RKKRRQRRR-G-(K).sub.15-G-RKKRRQRRR;
or a positively charged peptide having the amino acid sequence
YGRKKRRQRRR-G-(K).sub.15-G-YGRKKRRQRRR; or a positively charged
peptide having the amino acid sequences
RGRDDRRQRRR-G-(K).sub.15-G-RGRDDRRQRRR for use in the compositions
and methods of the invention.
[0047] Preferably the positively charged carrier includes
side-chain positively charged protein transduction domains or
positively charged efficiency groups in an amount of at least about
0.01%, as a percentage of the total carrier weight, preferably from
about 0.01 to about 50 weight percent, more preferably from about
0.05 to about 45 weight percent, and most preferably from about 0.1
to about 30 weight %. For positively charged protein transduction
domains having the formula -(gly).sub.n1-(arg).sub.n2, a preferred
range is from about 0.1 to about 25%.
[0048] In another embodiment, the backbone portion is a polylysine
and positively charged protein transduction domains are attached to
the lysine sidechain amino groups or to the C- or N termini. In
some preferred embodiments, the polylysine may have a molecular
weight that is at least 100, 200, 300, 400, 500, 600, 700, 800,
900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or
6000 D, and less than about 2,000,000, 1,000,000, 500,000, 250,000,
100,000, 75,000, 50,000, and 25,000 D. Within the range of 100 to
2,000,000 D, it is contemplated that the lower and/or upper range
may be increased or decreased, respectively, by 100, with each
resulting sub-range being a specifically contemplated embodiment of
the invention. In some exemplary embodiments, the polylysine has a
molecular weight from about 1,000 to about 1,500,000 D, from about
2,000 to about 800,000 D, or from about 3,000 to about 200,000 D.
In other exemplary embodiments, the polylysine has molecular weight
from about 100 to about 10,000 D, from about 500 to about 5,000 D,
from about 1,000 to about 4,000 D, from about 1,500 to about 3,500
D or from about 2,000 to about 3,000 D. Preferred is a polylysine
polypeptide having 10 to 20 lysines, more preferably, 15 lysines.
In some embodiments, the polylysine contemplated by this invention
can be any of the commercially available (Sigma Chemical Company,
St. Louis, Mo., USA) polylysines such as, for example, polylysine
having MW>70,000, polylysine having MW of 70,000 to 150,000,
polylysine having MW 150,000 to 300,000 and polylysine having
MW>300,000. The selection of an appropriate polylysine will
depend on the remaining components of the composition and will be
sufficient to provide an overall net positive charge to the
composition and provide a length that is preferably from one to
four times the combined length of the negatively charged
components. Preferred positively charged protein transduction
domains or efficiency groups include, for example,
-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly.sub.3Arg.sub.7) or
HIV-TAT.
[0049] In another preferred embodiment the positively charged
backbone is a polyalkyleneimine, non-limiting examples of which
include polyethyleneimine, polypropyleneimine, and
polybutyleneimine. In certain embodiments, the polyalkyleneimine
has a molecular weight of at least 100, 200, 300, 400, 500, 600,
700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,
5000, 5500, or 6000 D, and less than about 2,000,000, 1,000,000,
500,000, 250,000, 100,000, 75,000, 50,000, and 25,000 D. Within the
range of 100 to 2,000,000 D, it is contemplated that the lower
and/or upper range may be increased or decreased, respectively, by
100, with each resulting sub-range being a specifically
contemplated embodiment of the invention.
[0050] In other embodiments of this invention, the carrier is a
relatively short polylysine or polyethyleneimine (PEI) backbone
(which may be linear or branched) and which has positively charged
branching groups. Without wishing to be constrained by theory, it
is believed that such carriers are useful for minimizing
uncontrolled aggregation of the backbones and botulinum toxin in a
therapeutic composition, which causes the transport efficiency to
decrease dramatically. When the carrier is a relatively short
linear polylysine or PEI backbone, the backbone will have a
molecular weight of less than 75,000 D, more preferably less than
30,000 D, and most preferably, less than 25,000 D. When the carrier
is a relatively short branched polylysine or PEI backbone, however,
the backbone will have a molecular weight less than 60,000 D, more
preferably less than 55,000 D, and most preferably less than 50,000
D.
[0051] In one particularly interesting embodiment, the non-native
molecules are cationic peptides that have no inherent
botulinum-toxin-like activity and that also contain one or more
protein transduction domains as described herein. Without wishing
to be bound by any particular scientific theory, it is believed
that the peptides enhance tissue penetration of molecules
associated in complex after injection, while enhancing
stabilization of the botulinum toxin in skin and in vitro. It is
believed that the enhanced tissue penetration afforded by these
peptides in particular affords reduced antigenicity, a better
safety profile, enhanced potency, faster onset of clinical efficacy
or longer duration of clinical efficacy compared to conventional
commercial botulinum toxin complexes that are bound to exogenous
albumin (e.g., BOTOX.RTM. or) MYOBLOC.RTM..
[0052] In preferred embodiments, the concentration of positively
charged carriers in the compositions according to the invention is
sufficient to enhance the delivery of the botulinum toxin to
molecular targets such as, for example, motor nerve plates.
Furthermore, without wishing to be bound by theory, it is believed
that the penetration rate follows receptor-mediated kinetics, such
that tissue penetration increases with increasing amounts of
penetration-enhancing-molecules up to a saturation point, upon
which the transport rate becomes constant. Thus, in a preferred
embodiment, the amount of added penetration-enhancing-molecules is
equal to the amount that maximizes penetration rate right before
saturation. A useful concentration range for the positively charged
carrier (or carrier peptide) in the injectable compositions of this
invention is about 0.1 pg of carrier per Unit (U) of botulinum
toxin (0.1 pg/U) to about 1.0 mg per Unit (mg/U) of the botulinum
toxin as described herein. A useful concentration range for the
positively charged carrier (or carrier peptide) in the topical
compositions of the invention is about 1.0 pg/U to 0.5 mg/U of
botulinum toxin (amount of carrier/U of botulinum toxin). In other
embodiments, the positively charged carrier (or carrier peptide) is
present in the injectable compositions of the invention in the
range of, for example, 10 ng/U to 200 ng/U of botulinum toxin, or
in the range of 1 ng/U to 1000 ng/U of botulinum toxin; or in the
range of 0.1 ng/U to 10,000 ng/U of botulinum toxin. In some
embodiments, the amount of positively charged carrier (or carrier
peptide) to Units of botulinum toxin present in the compositions of
the invention is, by way of nonlimiting example, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, etc. ng of
carrier per Unit of botulinum toxin (ng/U). Preferably, the
botulinum toxin is of serotype A, and particularly, the 150 kD form
of serotype A botulinum toxin.
[0053] In general, methods and procedures for measuring the
activity of botulinum toxin, i.e., units (U) of botulinum toxin
activity, are known to and practiced by those having skill in the
art. Briefly, median lethality assays (LD.sub.50 assays) in mice
are conventionally used to estimate the number of units of
botulinum toxin with a high degree of precision. Doses of all
commercially available botulinum toxins are expressed in terms of
units of biologic activity. By way of example, one unit of
botulinum toxin corresponds to the calculated median
intraperitoneal lethal dose (LD50) in female Swiss-Webster mice.
See, Hoffman, R. O. et al., 1986, Int. Ophthalmol. Clin.,
26:241-50, as well as DePass, L. R., 1989, Toxicol. Letters,
49:159-170; and Pearce, L. B. et al., 1994, Toxicol. Appl.
Pharmacol., 128:69-77, which also describe lethality assays in the
art. More particularly, a suitable method for determining botulinum
toxin units for a botulinum toxin component of the compositions of
the invention is as follows: Forty-eight (48) female CD-1 mice
weighing 17-23 grams are randomly assigned to six doses of the test
article (1.54, 1.31, 1.11, 0.95, 0.80, and 0.68 U/0.5 mL), eight
(8) animals per dose group. The test article refers to the
botulinum toxin preparation or sample being assayed or tested. The
animals are housed eight per cage and are weighed within 24 hours
of dosing with the test article. On the day of dosing, the test
article is diluted to the appropriate concentrations in isotonic
saline (0.9% NaCl). Each animal is administered 0.5 mL of diluted
test article via intraperitoneal injection. After injection, mice
are returned to the cage and fatalities are recorded daily for
three days. Lethality is scored 72 hours post injection and the
results are analyzed by probit or logistic analysis to derive the
LD.sub.50 value relative to a reference standard that is assessed
using the same dosing regimen. By way of example, the reference
standard is a specifically qualified and calibrated lot of the same
composition of the invention that is used for comparison to derive
relative potency of the test article. The determined LD.sub.50
value is then corrected for the cumulative dilutions performed to
assign a relative potency value for the neat (undiluted) test
article.
[0054] Compositions of this invention are preferably in a form that
permits injection into the skin or epithelium of subjects or
patients, (i.e., humans or other mammals in need of the particular
treatment). The term "in need" is meant to include both
pharmaceutical or health-related needs (e.g., treating conditions
involving undesirable facial muscle spasms), as well as cosmetic
and subjective needs (e.g., altering or improving the appearance of
facial tissue). In preferred embodiments, the compositions are
prepared by mixing the botulinum toxin (either containing the
associated non-toxin proteins or reduced associated non-toxin
proteins) with the positively charged carrier, and usually with one
or more additional pharmaceutically acceptable carriers or
excipients. In their simplest form, they may contain an aqueous
pharmaceutically acceptable diluent, such as buffered saline (e.g.,
phosphate buffered saline). However, the compositions may contain
other ingredients typically found in injectable pharmaceutical or
cosmeceutical compositions, including a dermatologically or
pharmaceutically acceptable carrier, vehicle or medium that is
compatible with the tissues to which it will be applied. The term
"dermatologically or pharmaceutically acceptable," as used herein,
means that the compositions or components thereof so described are
suitable for use in contact with these tissues or for use in
patients in general without undue toxicity, incompatibility,
instability, allergic response, and the like. As appropriate,
compositions of the invention may comprise any ingredient
conventionally used in the fields under consideration, and
particularly in cosmetics and dermatology.
[0055] In terms of their form, compositions of this invention may
include solutions, emulsions (including microemulsions),
suspensions, gels, powders, or other typical solid or liquid
compositions used for injection to muscle and other tissues where
the compositions may be used. In preferred embodiments, the
compositions of the invention are present in low-viscosity, sterile
formulations suitable for injection with a syringe. As used herein,
the terms compositions and formulations are essentially
interchangeable when referring to the compositions and formulations
according to the present invention. The compositions of the
invention may be in the form of a lyophilized powder that is
reconstituted using a pharmaceutically acceptable liquid diluent
prior to injection. In certain embodiments, the lyophilized powder
is reconstituted with a liquid diluent to form an injectable
formulation with a viscosity of about 0.1 to about 2000 cP, more
preferably about 0.2 to about 500 cP, even more preferably about
0.3 to about 50 cP, and even more preferably about 0.4 to about 2.0
cP. The compositions of the invention may contain, in addition to
the botulinum toxin and positively charged carrier, other
ingredients typically used in such products, such as
antimicrobials, hydration agents, tissue bulking agents or tissue
fillers, preservatives, emulsifiers, natural or synthetic oils,
solvents, surfactants, detergents, gelling agents, antioxidants,
fillers, thickeners, powders, viscosity-controlling agents and
water, and optionally including anesthetics, anti-itch actives,
botanical extracts, conditioning agents, minerals, polyphenols,
silicones or derivatives thereof, vitamins, and
phytomedicinals.
[0056] The injectable compositions according to this invention may
be in the form of controlled-release or sustained-release
compositions which comprise botulinum toxin and positively charged
carrier encapsulated or otherwise contained within a material such
that they are released within the tissue in a controlled manner
over time. The composition comprising the botulinum toxin and
positively charged carrier may be contained within matrixes,
liposomes, vesicles, microcapsules, microspheres and the like, or
within a solid particulate material, all of which is selected
and/or constructed to provide release of the botulinum toxin over
time. The botulinum toxin and the positively charged carrier may be
encapsulated together (i.e., in the same capsule) or separately
(i.e., in separate capsules).
[0057] In embodiments, compositions of the invention comprise
liquid (aqueous) compositions (or formulations) comprising a
botulinum toxin as described herein, a positively charged carrier
(or peptide) as described herein, a non-reducing disaccharide or a
non-reducing trisaccharide, a non-ionic surfactant, and a
physiologically compatible buffer, which is capable of maintaining
a suitable pH, such as a pH in the range of pH 4.5 to pH 7.5, or pH
4.5 to pH 6.8, or pH 4.5 to pH 6.5. It is to be understood that a
suitable pH also includes the upper and lower pH values in the
range, e.g., a pH of 6.5 or a pH of 7.5. The concentration of the
non-reducing sugar in the liquid composition is in the range of 10%
through 40% (w/v) and the concentration of the non-ionic surfactant
is in the range of 0.005% through 0.5% (w/v). The liquid
compositions may be dried, preferably by lyophilization, to produce
stabilized solid compositions, which may thereafter be
reconstituted for use, for example, using sterile saline or other
known physiologically and pharmaceutically acceptable diluents,
excipients, or vehicles, especially those known for use in
injectable formulations. Preferably, the dried, e.g., lyophilized,
solid compositions are noncrystalline and amorphous solid
compositions, and may be in the form of powders, for example. Also,
preferably, the compositions of the invention do not include animal
protein-derived products, such as albumin. Compositions that are
suitable for the invention are also described in U.S. Application
Publication No. US 2010/0330123, the entire contents of which are
incorporated herein by reference. In particular embodiments the
compositions comprise botulinum toxin of serotype A. In other
particular embodiments, the compositions comprise botulinum toxin
of serotype A which has a molecular weight of 150 kDa.
[0058] In certain embodiments, the compositions of the invention
contain a non-reducing sugar, which is preferably a disaccharide,
non-limiting examples of which include trehalose, including its
anhydrous and hydrated forms, or sucrose, as well as combinations
thereof. In some embodiments, the hydrated form of trehalose,
trehalose-dihydrate, is preferable. In other embodiments, the
compositions contain a trisaccharide, a non-limiting example of
which is raffinose. In general, the concentration of the
non-reducing sugar, preferably a disaccharide, e.g., sucrose, in
the compositions of the invention are in the range of 10% to 40%
(w/v), preferably 10% to 25% (w/v), more preferably 15% to 20%
(w/v). In some preferred embodiments, the concentration of the
non-reducing sugar, preferably a disaccharide, e.g., sucrose, is
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% (w/v).
[0059] In general, the compositions of the invention may include
any non-ionic surfactant that has the ability to stabilize
botulinum toxin and that is suitable for pharmaceutical use. In
some embodiments, the non-ionic surfactant is a polysorbate, such
as, by way of nonlimiting example, polysorbate 20, polysorbate 40,
polysorbate 60, and polysorbate 80. In other embodiments, the
non-ionic surfactant is a sorbitan ester, non-limiting examples of
which include SPAN.RTM. 20, SPAN.RTM. 60, SPAN.RTM. 65, and
SPAN.RTM. 80. The non-ionic surfactants Triton.RTM. X-100 or NP-40
may also be used. In addition, a combination of the different
non-ionic surfactants may be used. In certain preferred
embodiments, the non-ionic surfactant is a polysorbate, a poloxamer
and/or a sorbitan; polysorbates and sorbitans are particularly
preferred. In embodiments, the non-ionic surfactant is present in
the compositions of the invention in the range of 0.005% to 0.5%,
or in the range of 0.01% to 0.2%, or in the range of 0.02% to 0.1%
or in the range of 0.05 to 0.08%, inclusive of the upper and lower
values. In addition, the compositions of the invention may contain
a non-ionic surfactant in the amount of 0.01%, 0.02%, 0.03%, 0.04%,
0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%,
0.14%, or 0.15%.
[0060] In general for the compositions of the invention, any
physiologically compatible buffer capable of maintaining the pH in
the above ranges is suitable for use. Non-limiting examples of such
buffers include salts of citric acid, acetic acid, succinic acid,
tartaric acid, maleic acid, and histidine. Non-limiting examples of
suitable buffer concentrations include buffer concentrations in the
range of 0.400% to 0.600%; 0.450% to 0.575%, or 0.500% to 0.565%.
The compositions of the invention may also comprise a mixture of
buffer salts, non-limiting examples of which include
citrate/acetate, citrate/histidine, citrate/tartrate,
maleate/histidine, or succinate/histidine. Accordingly, a
composition of the invention which provides a long duration effect
after treatment by a single injection includes a botulinum toxin,
such as botulinum toxin A or botulinum toxin A of 150 kDa MW, as
described herein, a positively charged carrier (or peptide) as
described herein, a non-reducing disaccharide, such as sucrose, a
non-ionic surfactant, such as polysorbate 20, polysorbate 40,
polysorbate 60, polysorbate 80, or a sorbitan ester, and a
physiologically compatible buffer, such as citric acid, acetic
acid, succinic acid, tartaric acid, maleic acid, and histidine,
which is capable of maintaining a suitable pH, such as a pH in the
range of pH 4.5 to pH 6.5 or in the range of pH 4.5. to pH 7.5, in
w/v amounts as described herein.
[0061] A particular composition of the invention is an
albumin-free, liquid (aqueous) composition which comprises a
botulinum toxin, preferably botulinum toxin of serotype A, or a
botulinum toxin A having a molecular weight of 150 kDa; a
positively charged carrier (e.g., peptide); a non-reducing
disaccharide or a non-reducing trisaccharide, preferably a
disaccharide, present in a range of 10% through 40% (w/v); a
non-ionic surfactant, preferably, a polysorbate or sorbitan ester,
present in the range of 0.005% through 0.5% (w/v); and a
physiologically compatible buffer, such as citric acid, acetic
acid, succinic acid, tartaric acid, maleic acid, or histidine,
present in the range of 0.400% to 0.600%; 0.450% to 0.575%, or
0.500% to 0.565%, for maintaining the pH between 4.5. and 7.5.
[0062] Botulinum toxin formulations according to the invention can
be delivered by injection (typically using a syringe) to muscles
underlying the skin, or to glandular structures within the skin, in
an effective amount to produce paralysis, produce relaxation,
alleviate contractions, prevent or alleviate spasms, reduce
glandular output, or other desired effects. Local delivery of the
botulinum toxin in this manner could afford dosage reductions,
reduce toxicity and allow more precise dosage optimization for
desired effects relative to injectable or implantable
materials.
[0063] The compositions of the invention are administered to
deliver an effective amount, preferably a therapeutically or
cosmetically effective amount, of the botulinum toxin. The term
"effective amount" or "therapeutically or cosmetically effective
amount" as used herein means an amount of a botulinum toxin as
defined above that is sufficient to produce the desired muscular
paralysis or other biological or aesthetic effect, but that
implicitly is a safe amount, i.e., one that is low enough to avoid
serious side effects. Desired effects include the relaxation of
certain muscles with the aim of, for instance, decreasing the
appearance of fine lines and/or wrinkles, especially in the face,
or adjusting facial appearance in other ways such as widening the
eyes, lifting the corners of the mouth, or smoothing lines that fan
out from the upper lip, or the general relief of muscular tension.
The last-mentioned effect, general relief of muscular tension, can
be effected in the face or elsewhere.
[0064] The compositions of the invention may contain an appropriate
effective amount of the botulinum toxin for application as a
single-dose treatment, or may be more concentrated, either for
dilution at the place of administration or for use in multiple
applications. Through the use of the positively charged carrier
this invention, a botulinum toxin can be administered by injection
to a subject for treating conditions such as wrinkles, undesirable
facial muscle or other muscular spasms, hyperhidrosis, acne, or
conditions elsewhere in the body in which relief of muscular ache
or spasms is desired. The compositions of the invention are
particularly suited for the treatment of fine lines, such as facial
fine lines, and glabellar lines, also known as "frown lines" in the
face of a subject. The botulinum toxin is administered by injection
to muscles or to other skin-associated or other target tissue
structures. The administration may be made, for example, to the
legs, shoulders, back (including lower back), axilla, palms, feet,
neck, face, groin, dorsa of the hands or feet, elbows, upper arms,
knees, upper legs, buttocks, torso, pelvis, or any other parts of
the body where administration of the botulinum toxin is
desired.
[0065] Administration of the injectable botulinum toxin-containing
compositions of this invention may also be carried out to treat
other conditions, including any condition for which prevention of
synaptic transmission of or release of acetylcholine would confer a
therapeutic benefit. For example, the conditions that may be
treated by the compositions according to the invention include,
without limitation, neurologic pain, migraine headache or other
headache pain, overactive bladder, rhinitis, sinusitis, acne,
dystonia, dystonic contractions (whether subjective or clinical),
hyperhidrosis (whether subjective or clinical), and hypersecretion
of one or more glands controlled by the cholinergic nervous system.
The compositions of this invention may also be used for reducing or
enhancing immune response, or treatment of other conditions for
which administration of botulinum toxin by injection has been
suggested or performed.
[0066] Most preferably, the compositions are administered by or
under the direction of a physician or other health care
professional. They may be administered in a single treatment or in
a series of treatments over time. In preferred embodiments, a
composition according to the invention is injected at a location or
locations where an effect associated with botulinum toxin is
desired. Because of its nature, the botulinum toxin preferably is
administered at an amount, application rate, and frequency that
will produce the desired result without producing any adverse or
undesired results. For instance, in certain embodiments, the
compositions of the invention are applied at a rate of from about 1
U to about 20,000 U, and more preferably from about 1 U to about
10,000 U botulinum toxin per cm.sup.2 of skin surface. Higher
dosages within these ranges may be employed, for example, in
situations where the botulinum toxin is administered in conjunction
with controlled release materials, as described herein. In certain
embodiments, the botulinum toxin formulations of the invention are
administered to provide 1 U to 400 U, preferably, 10 U to 300 U,
preferably 10 U to 350 U, preferably 30 U to 250 U and preferably
50 U to 200 U of botulinum toxin per injection. In other preferred
embodiments, the botulinum toxin-containing compositions of the
invention are administered by injection to provide 10 U to 100 U,
or 20 U to 100 U, or 20 U to 80 U, or 20 U to 60 U, or at least 20
U; at least 40 U, or at least 60 U, or 10 U, 20 U, 30 U, 40 U, 50
U, 60 U, 70 U, 80 U, 90 U, or 100 U of the botulinum toxin,
preferably botulinum toxin of serotype A, or botulinum toxin of
serotype A having a molecular weight of 150 kDa, to the subject
undergoing treatment. In embodiments, a single treatment with an
effective dose of the compositions of the invention affords an
effect of long duration such that during a course of treatment for
an indication treatable by botulinum toxin, for example, the
treatment of wrinkles, lines and furrows, a subject would require
fewer injections, and perhaps only a single injection, or series of
injections during a single treatment session, with a concomitant
effect that endures over extended periods of time, e.g., at least 6
months or greater than 6 months, namely, 6 months, 7 months, 8
months, 9 months, or longer, including 10 months. The longer
duration of action provides for longer intervals or time periods
between treatments where multiple treatments are used to maintain a
treatment goal or effect. In an embodiment, the longer duration of
effect of the composition following administration to, or dosing
of, an individual with a composition of the invention providing 20
U to 100 U, or 20 U to 60 U, or 20 U, 40 U, or 60 U of botulinum
toxin, for example, at least 6 months or greater than 6 months,
such as 7, 8, 9, or 10 months, including in between, is relative to
a duration of effect of a botulinum toxin-containing composition or
product that does not contain a positively charged carrier (or
peptide) according to the present invention. In some cases, a
composition or product containing botulinum toxin without a
positively charged carrier (or peptide) of the invention is
effective for less than 6 months, such as 3 or 4 months.
[0067] In certain embodiments, the compositions of the invention,
which comprise a botulinum toxin and a positively charged carrier
comprising a positively charged polymeric backbone with one or more
covalently attached positively charged efficiency groups as
described herein, are administered as a single injection to a
subject or patient in need thereof in an amount or at a dose which
provides 20 U to 60 U, or at least 20 U to at least 60 U of
botulinum toxin per injection for the treatment of wrinkles, lines,
or furrows, particularly in the face. According to the invention,
following a single injection in the treatment of a subject or
patient, a treatment effect, namely, the reduction of wrinkles,
lines, or furrows, such as glabellar lines, endures for several
weeks or months, for example, for at least 20 weeks, for at least
24 weeks, or for at least 6 months, or greater than 6 months, such
as 6, 7, 8, 9, or 10 months, or longer. In embodiments, the
botulinum toxin is of serotype A, B, C, D, E, F, or G. In an
embodiment, the botulinum toxin is of serotype A. In an embodiment,
the serotype A botulinum toxin has a molecular weight of 150 kDa.
In an embodiment, the serotype A botulinum toxin is in the form of
a higher molecular weight complex as described supra. In preferred
embodiments, the 150 kDa botulinum toxin or the higher molecular
weight forms of the toxin are in albumin-free formulations. In an
embodiment, the positively charged polymeric backbone is polylysine
or polyethyleneimine. In an embodiment, the one or more positively
charged efficiency groups include -(gly).sub.n1-(arg).sub.n2, in
which the subscript n1 is an integer of from 0 to 20, more
preferably 0 to 8, still more preferably 2 to 5, and the subscript
n2 is independently an odd integer of from about 5 to about 25,
more preferably about 7 to about 17, most preferably about 7 to
about 13. In some embodiments, the one or more positively charged
efficiency groups has the amino acid sequence
(gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20. In certain
preferred embodiments, p is one and q is zero or p is zero and q is
one. In other preferred embodiments, the subscripts p and q are
each independently integers of from 0 to 8, more preferably 0 to 5.
In a particular embodiment, the positively charged carrier has the
amino acid sequence RKKRRQRRRG-(K).sub.15-GRKKRRQRRR. In other
embodiments, the one or more positively charged efficiency groups
is attached to the positively charged backbone via either the
C-terminus or the N-terminus of the efficiency group, e.g., amino
acid sequence. In some embodiments, the one or more positively
charged efficiency groups are attached to either end, or both ends,
of the positively charged polylysine backbone of the positively
charged carrier. In particular embodiments, the positively charged
backbone is polylysine and the botulinum toxin is of serotype A. In
another particular embodiment, the serotype A botulinum toxin has a
molecular weight of 150 kDa, the positively charged backbone is
polylysine and the one or more covalently attached positively
charged efficiency groups has the amino acid sequence
(gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q, wherein the subscripts p and q
are each independently an integer of from 0 to 20, or are each
independently the values as set forth above; or the positively
charged carrier has the amino acid sequence
RKKRRQRRRG-(K).sub.15-GRKKRRQRRR. In embodiments, the composition
is administered by injection in an amount or dose that provides 20
U or at least 20 U; 30 U or at least 30 U; 40 U or at least 40 U;
50 U or at least 50 U; 60 U or at least 60 U; 70 U or at least 70
U; 80 U or at least 80 U; 90 U or at least 90 U; or 100 U or at
least 100 U of botulinum toxin per injection. Amounts or doses
between the foregoing amounts or doses are also contemplated, for
example, 25 U or at least 25 U; 35 U or at least 35 U; 45 U or at
least 45 U, and the like. In particular embodiments, the
composition is administered by injection as a single treatment dose
in an amount that provides 20 U; 40 U; or 60 U of botulinum toxin
and a response or effect, e.g., decrease in wrinkles and facial
lines, such as glabellar lines, is achieved and maintained for a
long duration, e.g., at least 20 weeks, at least 24 weeks, at least
6 months, or greater than 6 months, such as, for example, 6, 7, 8,
9, or 10 months, or longer. In a particular embodiment, a single
dose of a composition of the invention containing a positively
charged carrier as described and 150 kDa botulinum toxin A in a
dosage amount of 40 U provides a long duration effect in treating
glabellar lines, e.g., for at least 6 months and likely longer.
See, e.g., FIG. 4B.
[0068] Without wishing to be limiting, in a course of treatment,
the compositions of the invention may be administered at less
frequent intervals following an initial treatment dose based on the
extended duration of effect afforded by the therapeutically and
cosmetically effective doses of the compositions and methods of the
invention as described herein. For example, the compositions of the
invention may be administered (or dosed) to an individual in need
about twice per year (about every 6 months), or every 7 months, 8
months, 9 months, or 10 months, or longer, by the practice of the
methods of the invention. In a particular embodiment, an individual
is administered a dose of a composition of the invention twice per
year. A median duration between doses may be 6 months, at least 6
months, or greater than 6 months, depending on the therapeutic or
cosmetic treatment and/or the desire for treatment as determined by
the individual being treated. Thus, dosing of an individual with
the compositions of the invention may occur twice a year or longer
than twice a year, and for example, every 6, 7, 8, 9, or 10 months,
after an initial dose. A composition of the invention may be dosed
at the appropriate interval at 20 U to 100 U, more preferably, from
20 U to 60 U, and more preferably, 20 U, 40 U, or 60 U of botulinum
toxin in the composition.
[0069] This invention also contemplates the use of a variety of
delivery devices for injecting botulinum toxin-containing
compositions described herein across skin. Such devices may
include, without limitation, a needle and syringe, or may involve
more sophisticated devices capable of dispensing and monitoring the
dispensing of the composition, and optionally monitoring the
condition of the subject in one or more aspects (e.g., monitoring
the reaction of the subject to the substances being dispensed).
[0070] In some embodiments, the compositions can be pre-formulated
and/or pre-installed in a delivery device as such. This invention
also contemplates embodiments wherein the compositions are provided
in a kit that stores one or more components separately from the
remaining components. For example, in certain embodiments, the
invention provides for a kit that separately stores botulinum toxin
and the positively charged carrier for combining at or prior to the
time of application. The amount of positively charged carrier or
the concentration ratio of these molecules to the botulinum toxin
will depend on which carrier is chosen for use in the composition
in question. The appropriate amount or ratio of carrier molecule in
a given case can readily be determined, for example, by conducting
one or more experiments such as those described below.
[0071] In general, the invention also contemplates a method for
administering botulinum toxin (alternatively as botulinum toxin
complexes or reduced botulinum toxin complexes) to a subject or
patient in need thereof, in which an effective amount of botulinum
toxin is administered in conjunction with a positively charged
carrier, as described herein. By "in conjunction with" it is meant
that the two components (botulinum toxin and positively charged
carrier) are administered in a combination procedure, which may
involve either combining them prior to administration to a subject,
or separately administering them, but in a manner such that they
act together to provide the requisite delivery of an effective
amount of the therapeutic protein. For example, a composition
containing the positively charged carrier may first be administered
to the skin of the subject, followed by application a skin patch,
syringe, or other device containing the botulinum toxin. The
botulinum toxin may be stored in dry form in a syringe or other
dispensing device and the positively charged carrier may be
injected before application of the toxin so that the two act
together, resulting in the desired tissue penetration enhancement.
In that sense, thus, the two substances (positively charged carrier
and botulinum toxin) act in combination or perhaps interact to form
a composition or combination in situ. Accordingly, the invention
also includes a kit with a device for dispensing botulinum toxin
and a liquid, gel, or the like that contains the positively charged
carrier, and that is suitable for injection to the skin or target
tissue of a subject. Kits for administering the compositions of the
inventions, either under direction of a health care professional or
by the patient or subject, may also include a custom applicator
suitable for that purpose.
[0072] The compositions of this invention are suitable for use in
physiologic environments with pH ranging from about 4.5 to about
6.3, and may thus have such a pH. However, compositions having a pH
ranging from about 4.5 to about 7.5 are also embraced by the
invention as described herein. The compositions according to this
invention may be stored either at room temperature or under
refrigerated conditions.
[0073] In some embodiments, the patient to be treat is 65 years of
age, at least 65 years old, or over 65 years old. For example, the
patient may be 65, 66, 68, 70, 75, 80 years, or older.
[0074] It is understood that the following examples and embodiments
described herein are for illustrative purposes and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
[0075] All publications, patents, and published patent applications
cited herein are hereby incorporated by reference in their
entireties for all purposes.
EXAMPLES
Example 1
Duration of Local Muscle Paralysis in a Murine Model
[0076] This example compares the duration of local muscle paralysis
in mice injected with either RT003 or BOTOX.RTM.. RT003 is an
exemplary injectable formulation according to the invention that
contains type A botulinum toxin (purified to remove all endogenous
non-toxin proteins) and positively charged carrier with the
sequence RKKRRQRRRG-(K).sub.15-GRKKRRQRRR. BOTOX.RTM. also contains
type A botulinum toxin, but exogenous albumin is added to stabilize
the type A botulinum toxin molecule.
[0077] The muscle paralysis was measured using digit abduction
score (DAS) assay as reported by Aoki, K. R. in "A comparison of
the safety margins of botulinum neurotoxin serotypes A, B, and F in
mice", Toxicon 2001; 39(12):1815-1820. In the DAS assay, a mouse is
briefly suspended by its tail to cause a characteristic startle
response in which the mouse extends its hind limbs and abducts its
hind digits. The extent to which the mouse is able to exhibit this
startle response is scored on a five-point scale (from 0-4), with
zero representing a normal startle response and four representing
maximal reduction in digit abduction and leg extension. The scoring
is done by an observer with no knowledge of the extent to which the
subject mouse has been treated with neurotoxin. The baseline score
using the DAS assay was determined to be 0.4 for an untreated
population of animals.
[0078] The study reported in this example involved ten animals (5
mice in RT003 group and 5 mice in BOTOX.RTM. group). Each of the
animals was injected three times with the respective botulinum
toxin formulation (i.e., RT003 or BOTOX.RTM.), with a 40-day period
in between each dosing. After injection, the number of days that
all of the animals in each test group was above the 0.4 baseline of
the DAS assay was counted. The results, shown in FIG. 1, indicate
that the DAS assay score for the RT003-treated group stayed above
the 0.4 baseline value for 25, 22, and 21 days, following the
first, second, and third treatment, respectively. In contrast, the
DAS assay score for the BOTOX.RTM.-treated group stayed above the
0.4 baseline value for 11, 8, and 11 days, following the first,
second, and third treatment, respectively.
[0079] These DAS assay data indicate that local muscle paralysis
caused by the RT003 formulation lasts approximately twice as long
as the local muscle paralysis caused by BOTOX.RTM.. This result has
important implications for therapeutic uses of RT003 and other
injectable botulinum toxin-containing compounds according to the
invention. In particular, by using injectable compositions
according to the invention, one can significantly reduce the
frequency of follow-up injections required to maintain a particular
cosmetic or therapeutic effect caused by the botulinum toxin. In
turn, the reduced frequency of application can result in better
long-term efficacy, as the subject is less prone to develop
antibodies to the botulinum toxin.
Example 2
[0080] Injectable Botulinum Toxin Formulations with an Improved
Safety Profile
[0081] Over the last few decades, botulinum toxin has found use as
a therapeutic agent for treating a variety of conditions, including
wrinkles, hyperhidrosis, and muscle spasms. However, as botulinum
toxin is the most potent naturally occurring toxin known to humans,
improper administration of the toxin can be extremely dangerous.
For instance, accidental systemic delivery of botulinum toxin can
lead to paralysis, difficulty breathing, and even death. Moreover,
even if botulinum toxin were properly delivered to a localized
region of the body as a part of a therapeutic treatment, the toxin
has a natural tendency to diffuse over time, thereby increasing the
risk of unwanted paralysis in other parts of the body. For example,
when botulinum toxin is injected around the eyes to treat wrinkles,
it may diffuse to the muscles that control the movement of the
eyelids. If this happens, the eyelid muscles may become partially
paralyzed, leading to a well-known condition known as "eyelid
droop," in which the eyelid is partially closed and interferes with
normal vision.
[0082] One aspect of this invention is to provide injectable
botulinum toxin formulations with an improved safety profile
compared to currently available commercial botulinum toxin
formulations. In preferred embodiments, the injectable botulinum
toxin formulations have a reduced tendency to diffuse after
injection. In this way, certain preferred formulations of the
invention permit more accurate delivery of botulinum toxin,
dramatically reducing unwanted side effects associated with
uncontrolled local diffusion of botulinum toxin.
[0083] This example reports a comparative study of the tendency of
botulinum toxin in various formulations to diffuse following
injection. The study involved three botulinum toxin formulations:
(1) BOTOX.RTM.; (2) RT003, a buffered and stabilized solution
containing the 150 kD type A botulinum toxin molecule
non-covalently associated with a positively charged carrier having
the formula RKKRRQRRRG-(K).sub.15-GRKKRRQRRR; and (3) RTT150, which
is identical to the RT003 formulation, except that is does not
contain the positively charged carrier present in RT003.
[0084] The gastrocnemius muscle of each of the mice used in the
study was injected with one of the aforementioned botulinum toxin
formulations, either at the lateral-to-midline portion of the
muscle (FIG. 2A), or at the midline portion of the muscle (FIG.
2B). DAS assays were performed on each of the mice for four days
after injection with the botulinum toxin to determine whether the
botulinum toxin of the respective formulation exhibited any
tendency to diffuse from the gastrocnemius muscle toward the hind
paws of the mouse. From the DAS assays, any decreased ability of
the test animals to abduct their hind digits was interpreted as an
indication of botulinum toxin diffusion.
[0085] FIG. 3 shows the results of the DAS assays performed after
injecting the test animals with the different botulinum toxin
formulations as described above. Note that the digital abduction
scores are grouped into two clusters, corresponding to whether the
injection was at the midline or the lateral-to-midline portion of
the gastrocnemius muscle. The generally lower DAS scores for
midline injections, as compared to DAS scores for
lateral-to-midline injections, indicates that the degree of
paralysis in the hind paws of the test animals is generally less
following midline injection. Without wishing to be limited by
theory, it is believed that this behavior results from the greater
distance that botulinum toxin has to travel to reach the hind
digits of a test animal following midline injection, as compared to
lateral-to-midline injection. This greater required distance of
travel by the botulinum toxin is believed to decrease the
likelihood of paralysis of the hind digits.
[0086] FIG. 3 shows a digital abduction score of zero for all four
days following midline injection of the RT003 formulation. This
result indicates that the botulinum toxin in the RT003 formulation
stays localized in the midline portion of the gastrocnemius muscle
upon injection and that no paralysis-causing diffusion occurs on
the timescale of the experiment. By contrast, digital abduction
scores above the 0.4 DAS baseline are observed following injection
of the RTT150 and BOTOX.RTM. formulations, with the average DAS
score being higher for the BOTOX.RTM. formulation. The DAS results
for the RTT150 and BOTOX.RTM. formulations indicate that hind digit
paralysis of the test animals was observed after midline injection
of these formulations, with a greater degree of paralysis observed
after the injection of the BOTOX.RTM. formulation. These data
suggest that the botulinum toxin molecules in the RTT150 and
BOTOX.RTM. formulations are capable of locally diffusing after
injection, with a greater degree of local diffusion for the
botulinum toxin molecules in the BOTOX.RTM. formulation.
[0087] FIG. 3 also shows that hind digit paralysis is observed for
all test animals following lateral-to-midline injection,
irrespective of the specific botulinum toxin formulation. As
discussed above, this greater degree of paralysis following
lateral-to-midline injection, as compared to midline injection, is
believed to relate to a shorter travel distance for the botulinum
toxin to the hind paws of the test animals. However, while all
three botulinum toxin formulations exhibit paralysis-causing
diffusion following lateral-to-midline injection, the degree of
paralysis in test animals injected with RT003 is less, on average,
than the degree of paralysis observed for the RTT150 and BOTOX.RTM.
formulations during the timescale of the experiment. Thus, the DAS
assay data corresponding to lateral-to-midline injection is
qualitatively similar to that for midline injection in that it
shows a decreased tendency for local diffusion of botulinum toxin
for the RT003 formulation, as compared to RTT150 and
BOTOX.RTM..
[0088] A comparison of the local diffusion rate following midline
injection and lateral-to-midline injection can be made by
considering a parameter called the "diffusion index", which is
defined according to Equation (1):
diffusion index = midline digital abduction score lateral - to -
midline digital abduction score .times. 100. ( 1 ) ##EQU00001##
[0089] Since digital abduction scores can range from 0 to 4, and
lateral-to-midline digital abduction scores are expected to be
higher than midline digital abduction scores (as discussed above),
diffusion index values will typically range from 0 to 100. A
diffusion index value that approaches 100 indicates that the ratio
of the midline and lateral-to-midline digital abduction scores
approaches unity. This may occur if the rates of diffusion
following injection are sufficiently high that the diffusion times
for the botulinum toxin to reach and to paralyze the hind digits of
the test animal following midline and lateral-to-midline injection
are comparable or nearly the same. At the other extreme, diffusion
index values that approach zero indicate that the ratio of the
midline and lateral-to-midline digital abduction scores is
approaching zero. This may occur if diffusion of the botulinum
toxin following midline injection is so low that it is insufficient
to cause paralysis in the hind digits of the test animals, even
though paralysis is observed following lateral-to-midline
injection.
Table 1 below shows diffusion index values calculated using digital
abduction scores following midline or lateral-to-midline injection
of BOTOX.RTM., RT003, and RTT150, as reported in the experiment
corresponding to FIG. 3. On the timescale of the experiment, the
diffusion index values corresponding to injection of the BOTOX.RTM.
formulation are higher than the values observed for the RTT150 and
RT003 formulations. This indicates that, for injection of the
BOTOX.RTM. formulation, the ratio of the midline and
lateral-to-midline digital abduction scores are closer to unity,
compared to the ratios observed for the RTT150 and RT003
formulations. Since botulinum toxin must diffuse further to cause
hind-digit paralysis of a test animal following midline injection,
the observation that the ratio of the midline and
lateral-to-midline digital abduction scores following BOTOX.RTM.
injection is closer to unity suggests that the botulinum toxin
diffusion rate following midline injection of BOTOX.RTM. is fairly
substantial relative to the rate following lateral-to-midline
injection. In other words, the increased diffusion path length
associated with midline injection is less of a barrier to causing
hind-digit paralysis.
[0090] In contrast, the diffusion index values for RT003 are all
zero on the four-day timescale of the experiment. This result
indicates that no paralysis-inducing diffusion is observed
following midline injection of RT003. In other words, the RT003
formulation, which contains the type A botulinum toxin molecule
non-covalently associated with a positively charged carrier,
permits enhanced localization injected type A botulinum toxin. In
this way, the RT003 formulation affords an improved safety profile
compared to that of the BOTOX.RTM. formulation and minimizes
unwanted paralysis.
[0091] The observed diffusion index values for RTT150, while not
zero as in the case of RT003, are still less than those observed
for the BOTOX.RTM. formulation. See, Table 1. This result indicates
that enough botulinum toxin diffusion occurs to produce observable
hind digit paralysis on the four-day timescale of the experiment,
but that the time required for paralysis-causing diffusion of
botulinum toxin is relatively longer following midline
injection.
TABLE-US-00001 TABLE 1 Botulinum toxin diffusion index measurements
for RTT150, BOTOX .RTM. and RT003 Days Post Treatment 0 1 2 3 4
BOTOX .RTM. NA 42 38 38 9 RT003 NA 0 0 0 0 RTT150 NA 20 20 27
17
Example 3
[0092] Injectable Botulinum Toxin Formulations with Reduced
Tendency to Generate Antibodies
[0093] When botulinum toxin is periodically injected into a patient
to treat an unwanted condition such as wrinkles, it is often
observed that efficacy of the botulinum toxin decreases with
successive injections, even though the duration of the effects of
the botulinum toxin may remain the same. This phenomenon is
believed to be the result of the formation of antibodies to the
botulinum toxin by the immune system of the patient. From a
treatment perspective, the formation of antibodies to botulinum
toxin by the patient is undesirable, because increasingly larger
doses of botulinum toxin are then required to achieve the same
effect, which presents serious issues related to both safety and
cost.
[0094] In certain embodiments, this invention provides injectable
botulinum toxin formulations that have a decreased tendency to
induce antibody formation, as compared to currently available
commercial injectable botulinum toxin formulations. Thus, in these
embodiments, botulinum toxin formulations help to minimize the risk
associated with botulinum toxin injection by permitting one, over
time, to use less toxin to achieve the same effect.
[0095] In this example, the DAS assay data obtained after repeated
RT003 and BOTOX.RTM. injections as described in Example 2 are
analyzed as a function of time to determine how the efficacy of
these two formulations changes upon repeated administration to the
same test animals. Generally, after repeated administration of
either formulation, the duration of effects associated with
botulinum toxin were the same. However, the degree of muscle
paralysis upon repeated administration varied depending on the
formulation. To quantify the change in the degree of muscle
paralysis, the percent change in the digital abduction scores
following injection of either RT003 or BOTOX.RTM. was determined
according to Equation (2):
% change in DAS = DAS for nth treatment - DAS for first treatment
DAS for first treatment .times. 100 % . ( 2 ) ##EQU00002##
[0096] Since the numerator of Equation (2) is the difference
between the measured digital abduction scores for the n.sup.th and
the first treatment, the percent change in DAS will be negative if
the digital abduction score measured for the n.sup.th treatment is
less than the digital abduction score measured for the first
treatment. In other words, the percent change in DAS is negative
when less paralysis is observed after the nth treatment, as
compared to the first treatment. Table 2 shows the percent change
in the measured DAS values following repeated administration of
RT003 and BOTOX.RTM. formulations according to the procedure
described in Example 2.
TABLE-US-00002 TABLE 2 Percent Change in DAS Value after Repeated
Administration of RT003 and BOTOX .RTM. 1st 1st 2nd treatment
retreatment retreatment RT003 0% 0% -30% BOTOX .RTM. 0% -44%
-67%
[0097] As indicated in Table 2, after the first retreatment, the
percent change in the digital abduction score was -44% for the
BOTOX.RTM. formulation, which suggests a substantial drop in the
efficacy. In contrast, the percent change in the digital abduction
score for the RT003 formulation was zero, indicating that the DAS
score after the second retreatment was the same as after the
initial administration and first retreatment. This result indicates
that the degree of paralysis observed after the first retreatment
of RT003 is the same as the degree of paralysis following the first
treatment and that negligible formation of neutralizing antibodies
occurred in the test animals even after the first retreatment.
After the 2nd retreatment of RT003 and BOTOX.RTM., the calculated
percent changes in DAS values were negative for both formulations,
although the magnitude of the percent change in DAS values for the
RT003 formulation was half of the value determined for BOTOX.RTM..
The larger and negative percent change in DAS values observed for
BOTOX.RTM. suggest that the test animals had a higher rate of
antibody generation to BOTOX.RTM., as compared to RT003. Thus,
these data indicate that formulations contemplated by this
invention, such as RT003, may have a lower tendency to induce the
formation of antibodies that neutralize the effect of botulinum
toxin. Accordingly, this result suggests that by using formulations
contemplated by this invention, one can, over time, use less
botulinum toxin to achieve the same therapeutic effect.
Example 4
Injectable Botulinum Toxin Formulations With Improved Stability
[0098] This example demonstrates that the positively charged
carrier molecules used in the injectable botulinum toxin
formulations of the invention not only enhance the safety profile
of the formulations (Example 2), but also improve their stability.
Table 3 shows the results of aging experiments wherein the RT003
and RTT150 formulations are aged at 4.degree. C. (RT003 only) and
at 40.degree. C. (both RT003 and RTT150) for various time
intervals. After aging at the specified temperatures for the
specified times, the potency of the RT003 and RTT150 formulations
were measured via a series of mouse IP LD.sub.50 assays. The
results, summarized in Table 3, indicate that the potency of RT003
is essentially unchanged following aging at 4.degree. C., even
after six months. Furthermore, the potency of the RT003
formulation, as measured by the formulation's ability to kill the
target animals in a mouse IP LD.sub.50 assay, decreases only
slightly even if the RT003 formulation is aged at elevated
temperature (40.degree. C.) for six months. By contrast, the RTT150
formulation exhibited a significant decrease in potency following
only one month of aging at 40.degree. C. Since the RT003 and RTT150
formulations are identical, with the exception that the RT003
formulation also contains a positively charged carrier molecule
having the formula RKKRRQRRRG-(K).sub.15-GRKKRRQRRR, these data
indicate that the positively charged carrier molecule improves the
stability of the botulinum toxin in the RT003 formulation.
TABLE-US-00003 TABLE 3 Results of Mouse IP LD.sub.50 Assays
following Aging of RT003 and RTT150 At Various Conditions Condition
Time (.degree. C.) (months) % Target RT003 4 0 100% 4 6 118% 40 6
93% RTT150 40 1 <50%
Example 5
Injectable Botulinum Toxin Formulation Showing Long-Lasting
Duration Effects in the Treatment of Glabellar Lines
[0099] This Example describes a clinical study and interim analysis
of results at week 24 to evaluate the safety, efficacy and duration
of effect of an injectable composition of the invention containing
botulinum toxin A and a positively charged carrier comprising a
positively charged polylysine polypeptide having covalently
attached one or more positively charged efficiency groups, called
RT002. The RT002 product is an injectable formulation, which
contains the 150 kD subtype A botulinum toxin molecule, which is
not covalently associated with a positively charged carrier peptide
having the formula RKKRRQRRRG-(K).sub.15-GRKKRRQRRR, and which does
not contain accessory proteins or animal-derived components, used
in the study for the treatment of moderate to severe glabellar
lines.
[0100] The clinical study was a phase 2, randomized, double blind,
dose ranging, active and placebo-controlled, multi-center study
designed and conducted to evaluate the safety and efficacy and
duration of effect of a single (one-time) treatment by injection of
RT002 for the temporary improvement in the appearance of glabellar
lines in adults. Three doses, 20 U, 40 U and 60 U, of RT002 were
evaluated compared to an active, i.e., VISTABEL.RTM./BOTOX.RTM. (20
U dose by intramuscular injection) and a placebo control
(intramuscular injection). The injection treatment was a single
intramuscular injection. The duration of effect of a single
treatment of RT002 at the three dosage levels versus
VISTABEL.RTM./BOTOX.RTM. Cosmetic was also assessed.
[0101] The RT002 product is composed of purified 150 kDa botulinum
neurotoxin, referred to as RTT150, formulated in a lyophilized
powder. In nonclinical studies, RT002 has been shown to exhibit
less diffusion than other forms of botulinum neurotoxin A (BoNTA)
and may offer more control of effect at target sites with less side
effects due to distant spread of toxin into neighboring muscles. In
addition, the RT002 additive-free botulinum toxin type A
formulation has the ability to afford less immunogenic potential
due to the absence of non-active proteins present in the
formulation. In addition, RT002 was well tolerated after repeat
dose intramuscular administration of up to 50 U/kg in rats.
[0102] Dosing Regimen and Injection Technique:
[0103] The dosing regimen of RT002 for this study was a single
treatment of either RT002 (20 U, 40 U, or 60 U), placebo, or
VISTABEL.RTM./BOTOX.RTM., which was dosed at 20 U per subject, as a
0.1 mL intramuscular injection into each of 5 injections sites on
the forehead (0.5 mL total), between the eyebrows, of the subject
undergoing treatment. All treatments were intramuscular injections
administered by a trained physician. More specifically, study
subjects received a single treatment of 0.1 mL per injection to
five injection sites: two injections into each corrugator muscle,
and one injection in the procerus muscle. Investigators, site
staff, subjects, and the sponsor were blinded to the treatment
group assignments. Approximately 250 adult, female and male
subjects, 30 to 65 years of age and in good general health, with
moderate to severe glabellar lines at entry, were enrolled in the
study.
[0104] Glabellar facial lines arise from the lateral corrugator and
vertical procerus muscles in the face. These can be readily
identified by palpation of the muscle mass while having the patient
frown maximally. The corrugator depresses the skin creating a
vertical line, i.e., a furrow, surrounded by ridges of tensed
muscle (i.e., frown lines). Because the location, size and use of
the muscles vary markedly among individuals, physicians
administering injectable botulinum toxin must understand the
relevant anatomy of the area involved and any alterations to the
anatomy due to prior surgical procedures. In order to reduce the
risk of ptosis, the following steps are optimally performed: (i)
injection of or near the levator palpebrae superioris should be
avoided, particularly in patients with larger brow depressors; (ii)
medial corrugator injections should be at least 1 centimeter above
the bony supraorbital ridge; (iii) it should be ensured that the
injected volume/dose is accurate; and (iv) toxin should not be
injected closer than 1 centimeter above the central eyebrow.
Botulinum toxin is injected by applying finger pressure on the
superior medial orbital rim while advancing the needle through the
skin into the underlying muscle.
[0105] For the study, the severity of a subject's glabellar lines
was assessed by the Investigator and the subject. For the
Investigator assessment, an Investigator Global Assessment-Facial
Wrinkle Severity (IGA-FWS) rating score system was used as follows:
an IGA-FWS rating score of (0) indicated no facial wrinkle
severity; an IGA-FWS rating score of (1) indicated mild facial
wrinkle severity; an IGA-FWS rating score of (2) indicated moderate
facial wrinkle severity; and an IGA-FWS rating score of (3)
indicated severe facial wrinkle severity. As appreciated by the
skilled practitioner, a photo guide exhibits the grades of wrinkle
severity used for Investigator and reference.
[0106] Patient Facial Wrinkle Severity (PFWS) Assessment:
[0107] A Patient Facial Wrinkle Severity (PFWS) was used for a
subject's assessment of his/her facial wrinkle severity. Subjects
completed the Patient Facial Wrinkle Severity (PFWS) at maximum
frown to assess the severity of the glabellar lines at the
Screening Visit, Treatment Visit (Day 0) pre-treatment, Follow-up
Visits (Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32), and End-of-Study
Visit (Week 24, 28, 32 or 36, as appropriate) or Early
Discontinuation Visit, if applicable. The assessment form was
provided directly to the subject to complete while reviewing the
glabellar lines using a supplied handheld mirror. The PFWS rating
score system was as follows: a PFWS rating score of (0) indicated
no wrinkle severity, with associated description of "no wrinkles;"
a PFWS rating score of (1) indicated mild wrinkle severity, with
associated description of "very shallow wrinkles;" a PFWS rating
score of (2) indicated moderate wrinkle severity, with associated
description of "moderate wrinkles;" and a PFWS rating score of (3)
indicated severe wrinkle severity, with associated description of
"deep wrinkles." In accordance with the study, an IGA-FWS and a
PFWS rating of (2) moderate or (3) severe for a subject's glabellar
lines were required for a subject to be enrolled in the study.
[0108] Subjects were randomized 1:1:1:1:1 to one of the treatments
presented in Table 4 below.
TABLE-US-00004 TABLE 4 Description of Treatment Groups Treatment
Group Test Article and Dose No. of Subjects 1 RT002 20 U 50 2 RT002
40 U 50 3 RT002 60 U 50 4 Placebo 50 5 Active comparator 50
(VISTABEL .RTM./BOTOX .RTM. 20 U)
[0109] Subjects enrolled in the study had screening and treatments
visits and follow-up safety and efficacy evaluations throughout the
study for up to 36 weeks. A subject diary was provided for the
initial 2-week period to document onset of treatment response.
Subjects were evaluated with a phone call at Week 1 and during
visits at Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32 and 36 of the
study. All subjects were followed for at least 24 weeks
post-treatment. If the subject's Investigator Global
Assessment-Facial Wrinkle Severity (IGA-FWS) score at maximum frown
returned to baseline between the 24 week and 36 week visits, the
visit at which that score was recorded was considered the
End-of-Study Visit for the subject.
[0110] The study duration was up to 38 weeks on study, including a
screening period of up to two weeks followed by a single treatment,
and a follow-up period of up to 36 weeks post-treatment. All
subjects were followed for at least 24 weeks post-treatment.
Injection sites were evaluated at the Screening Visit, Treatment
Visit (Day 0), pre- and post-treatment (to determine if there was
an immediate reaction to the investigational product), Follow-up
Visits (Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32), and End-of-Study
Visit (Week 24, 28, 32 or 36, as appropriate) or Early
Discontinuation Visit, if applicable. The assessment was done as a
global evaluation of the 5 injection sites and evaluated erythema,
edema, burning or stinging sensation, and itching, as described by
the subject.
[0111] In addition, cranial nerves II-VII were evaluated by the
Investigator at the Treatment Visit (Day 0) pre-treatment, at
Follow-up Visits (Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32) and at the
End-of-Study Visit (Week 24, 28, 32, or 36, as appropriate) or
Early Discontinuation Visit, if applicable. Scores for the cranial
nerve assessments were captured as follows: a rating of (1)
corresponded to "Normal"; a rating of (2) corresponded to
"Abnormal, not clinically significant;" a rating of (3)
corresponded to "Abnormal, clinically significant;" a rating of (4)
corresponded to "Not assessed." For these assessments, cranial
nerve II is the optic nerve; cranial nerve III is the oculomotor
nerve; cranial nerve IV is the trochlear nerve; cranial nerve V is
the trigeminal nerve; cranial nerve VI is the abducens nerve and
cranial nerve VII is the facial nerve. The Regional House-Brackmann
Facial Nerve Grading System (Yen, T. L. et al., 2003, Otol.
Neurotol., 24(1):118-122) was designed to evaluate synkinesis and
the four major branches of the facial nerve (VII) that innervates
target and adjacent musculature. The Investigator evaluated
functionality of the facial nerve (VII) at the Treatment Visit (Day
0) pre-treatment, Follow-up Visits (Weeks 2, 4, 8, 12, 16, 20, 24,
28, 32) and End-of-Study Visit (Week 24, 28, 32, or 36, as
appropriate) or Early Discontinuation Visit, if applicable.
[0112] Facial muscle strength was evaluated using the Medical
Research Council Scale for Assessment of Muscle Power (MRC). The
MRC is a reliable and validated scale for assessing muscle weakness
and aids the investigation of peripheral nerve injuries
(Paternostro-Sluga, 2008). The orbicularis oculi (eyelid), lateral
brow elevators, and lateral orbicularis zygomaticus muscles on each
side of the face were evaluated. In the MRC Scale for Muscle Power
Assessment, a rating of (0) corresponds to "no movement;" a rating
of (1) corresponds to "flicker perceptible in the muscle;" a rating
of (2) corresponds to "movement only is gravity is eliminated;" a
rating of (3) corresponds to "can move limb against gravity;" a
rating of (4) corresponds to "can move against gravity and some
resistance exerted by examiner; and a rating of (5) corresponds to
"normal power."
[0113] Distant spread of toxin queries were conducted with subjects
at the Treatment Visit (Day 0) post-treatment, Follow-up Phone Call
(Week 1), Follow-up Visits (Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32),
and End-of-Study Visit (Week 24, 28, 32, or 36, as appropriate) or
Early Discontinuation Visit, if applicable. In addition, adverse
events (AEs) were also evaluated at these same time points. Without
wishing to be limiting, examples of AEs include double vision,
eyelid paralysis, muscle weakness, extreme tiredness and difficulty
swallowing, breathing and speaking.
[0114] Efficacy assessments included Investigator assessment of
glabellar line severity and glabellar line improvement scales,
subject assessment of glabellar line severity and improvement
including subject questionnaires, and onset of effect evaluated by
subject diary. Efficacy assessments were conducted with the subject
in a sitting position. In order to have consistent eye positioning
during the assessment, the Investigator asks the subject to focus
on a fixed point in the examination room. The assessment should be
conducted in a room with good overhead lighting (an exam light
should not be used) or natural light from a window (but not direct
sunlight). At each clinic visit, the visual appearance (at maximum
frown and at rest after maximum frown) of the glabellar lines was
assessed by the Investigator using a fit-for-purpose 4 point
IGA-FWS scale/rating score for Facial Wrinkle Severity Score, as
follows: a rating score of (0) corresponded to no facial wrinkles;
a rating score of (1) corresponded to mild facial wrinkles; a
rating score of (2) corresponded to moderate facial wrinkles; and a
rating score of (3) corresponded to severe facial wrinkles. The
assessment represented wrinkle severity at each given time-point
and was not based on a comparison to the pre-treatment level.
Assessments were optimally completed by the same Investigator and
as close as possible to the same time of day at each visit. In an
effort to standardize the rating of wrinkle severity across
Investigators, a set of training photographs exhibiting the grades
of wrinkle severity was used for Investigator training. A photo
guide was also provided to each study center to assist in the
Investigator's assessment.
[0115] Patient Global Aesthetic Improvement Scale (GAIS):
[0116] The Investigator and subject assessed the visual appearance
(at maximum frown and at rest after maximum frown) of the glabellar
line improvement from the baseline condition using the 7 point
severity Patient Global Aesthetic Improvement Scale (GAIS) shown in
Table 5 below. Study subjects completed the Patient Global
Aesthetic Improvement Scale (GAIS) at maximum frown and at rest
after maximum frown, to assess the visual appearance of the
glabellar line improvement from the baseline condition at Follow-up
Visits (Weeks 2, 4, 8, 12, 16, 20, 24, 28, 32), and End-of-Study
Visit (Week 24, 28, 32, or 36, as appropriate) or Early
Discontinuation Visit, if applicable. The GAIS assessment form was
provided directly to the subject to complete while reviewing the
treated area using a supplied handheld mirror. Subjects with
contact lenses optimally viewed their glabellar lines while wearing
their contacts. Subjects wearing glasses were advised to view their
glabellar lines without glasses if possible. If glasses were needed
for the subject to see their glabellar lines, then glasses were
worn for the assessment. The subject assessment was completed
before the Investigator completed the IGA-FWS assessment.
TABLE-US-00005 TABLE 5 Global Aesthetic Improvement Scale Rating
Score Wrinkle Improvement -3 Very Much Worse -2 Much Worse -1 Worse
0 No Change 1 Improved 2 Much Improved 3 Very Much Improved
[0117] At each clinic visit, the subject assessed the visual
appearance (at maximum frown) of the glabellar lines using the
following fit-for-purpose 4 point scale for subject's assessment of
Patient-Facial Wrinkle Severity (Table 6 below). The assessment
form was provided directly to the subject to complete while
reviewing the glabellar treatment area using the supplied handheld
mirror. As for the above GAIS assessment, subjects with contact
lenses optimally viewed their glabellar lines while wearing their
contacts. Subjects wearing glasses were advised to view their
glabellar lines without glasses if possible. If glasses were needed
for the subject to see their glabellar lines, then glasses were
worn for the assessment. The subject assessment was completed
before the Investigator completes the IGA-FWS assessment. The
assessment represented wrinkle severity at each given time-point
and was not based on a comparison to the pre-treatment defect
level. Assessments were optimally completed by the subject as close
to the same time as possible at each visit.
TABLE-US-00006 TABLE 6 Patient-Facial Wrinkle Severity (PFWS)
Rating Score Wrinkle Severity Description 0 None No wrinkles 1 Mild
Very shallow wrinkles 2 Moderate Moderate wrinkles 3 Severe Deep
wrinkles
[0118] Additional subject assessments during the study included a
rating of the importance of the duration of effect when choosing an
aesthetic treatment (provided at the Treatment Visit (Day 0); a
rating of subjects' satisfaction with the treatment results (at the
Week 4 visit), in the form of a questionnaire to rate their
satisfaction with the treatment results--the subjects were asked
how satisfied or dissatisfied they were with the appearance of the
treated area of the face; and a rating of their satisfaction with
the duration of the treatment effect (at the End-of-Study Visit
(Week 24, 28, 32, or 36, as appropriate) or Early Discontinuation
Visit, if applicable.
[0119] Digital photographs of the treatment area were taken at the
Treatment Visit (Day 0) pre-treatment, Follow-up Visits (Weeks 2,
4, 8, 12, 16, 20, 24, 28, 32), and at End-of-Study Visit (Week 24,
28, 32, or 36, as appropriate) or Early Discontinuation Visit.
Digital photographs were taken in a controlled and standardized
manner. Reference photographs and appropriate training were
provided to site staff and Investigators. Subjects optimally did
not wear eye or facial make-up of any kind. In order to minimize
light reflection from the skin, treated areas were blotted with an
alcohol pad and allowed to dry to remove skin oil prior to taking
any photographs. Photographs included the subject's frontal view at
maximum frown and at rest after maximum frown.
[0120] Statistical Analysis:
[0121] All statistical programming and analyses were performed
using SAS version 9.3 or higher. As this study was not powered to
detect any statistically significant differences between treatment
groups at the 0.05 level, the p-value obtained from various tests
described below are expected to establish statistical trending. No
adjustments were made for multiplicity of testing. Demographic and
baseline characteristics were summarized for the intent-to-treat
(ITT), per-protocol (PP) and safety populations. Descriptive
statistics were provided for all efficacy variables at all
time-points by treatment group as well as by treatment group and
geography/country. Efficacy analyses were performed for the ITT and
PP populations. Safety analyses were performed on the safety
population.
[0122] Populations:
[0123] All subjects who were randomized and received treatment were
included in the intent-to-treat (ITT) population. All subjects who
were randomized, received treatment, and had provided at least one
post-treatment safety assessment were included in the Safety
Population. The Per Protocol (PP) population included subjects from
the ITT population who complete the 24-week evaluation without a
major protocol violation. Subjects were excluded from the PP
population for any of the following reasons: (i) the subject
violated inclusion/exclusion criteria; (ii) the subject missed the
week 24 visit; (iii) the subject used a prohibited medication; (iv)
the subject's Week 24 visit was .+-.5 days off-schedule (outside of
allowed variation in scheduled visit days).
[0124] For safety groups and efficacy comparisons, subjects were
randomized into 5 treatment groups (RT002 20 U; RT002 40 U; RT002
60 U; Placebo; Active Comparator). The primary efficacy comparisons
were performed between each RT002 dose and active comparator; each
RT002 dose and placebo; as well as active comparator to placebo. A
risk-to-benefit ratio was evaluated to examine trends in favor of
at least one of the RT002 doses versus active comparator in key
study evaluations (proportion of responders at month 6 and duration
of response measured up to 36 weeks; frequency of AEs).
[0125] Within each treatment group, the missing scores for IGA-FWS,
PFWS, and GAIS for the ITT population were imputed by Markov Chain
Monte Carlo (MCMC) multiple imputation for analyses based on
proportion of responders. The sensitivity analysis for the primary
endpoint was performed using imputations based on the last
observation carried forward method.
[0126] Descriptive statistics were used to summarize demographic
characteristics (e.g., age, gender, race, etc.) and background
characteristics (e.g., IGA-FWS, PFWS, etc.). Past or ongoing
medical history, study visit compliance, and prior and concomitant
medication usage were summarized for all subjects and presented in
a listing by subject.
[0127] Efficacy:
[0128] For efficacy, primary clinical efficacy were assessed by
blinded evaluator who graded the severity of the subject's
glabellar lines at maximum frown using the IGA-FWS. A responder is
defined as a subject who has a one point or greater improvement in
IGA-FWS versus baseline and who has not returned to baseline
IGA-FWS at the time point of evaluation. For the primary analysis
purposes, the Proportion of Responders was compared between each
RT002 dose and active comparator at Week 24. Each RT002 treatment
group was compared separately to placebo and active comparator.
Active comparator was compared to placebo at each visit also.
Comparisons were made with Cochran-Mantel-Haenszel (CMH) tests
stratified by baseline IGA-FWS.
[0129] For the primary analysis purposes, Duration of Response was
compared between each of the RT002 doses and active comparator
using the Kaplan-Meier method. The duration of response was
measured from the time of injection to the time point when a
subject reverted to his/her baseline severity as measured by
Blinded Investigator based on IGA-FWS. If the subject did not
achieve a one point improvement from baseline by IGA-FWS on or
before Week 4, the duration of response was considered zero. If
subject achieved at least a 1 point improvement based on IGA-FWS on
or before week 4, but did not revert to his/her baseline by Week 36
(last time point), such a subject was censored at Week 36 (date of
last evaluation) for the analysis. The log-rank test was used to
compare duration of response between RT002 and active comparator. A
Risk to Benefit Ratio computed for each treatment group was equal
to the sum of the number of treatment related adverse events
divided by the sum of the duration of response days for the
subjects in the treatment group. If a subject achieved at least 1
point improvement based on IGA-FWS on or before week 4 but did
revert to his/her baseline by Week 36 (last time point), then
his/her contribution to the benefit sum was the number of days
between baseline and the last visit day.
[0130] For secondary analyses, secondary endpoints used are defined
as follows:
[0131] (1) Proportion of Responders at Week 2 and at Weeks 4-36
with the emphasis on Week 12 and Week 24 evaluations. The
comparisons between treatment groups were based on the CMH test
stratified by baseline severity of the variable analyzed where
possible. Each treatment group was compared to placebo and
separately compared to active comparator for those subjects who had
a baseline severity which could possibly permit the required
improvement for success. Active comparator was also compared to
placebo at each visit.
Responders were evaluated based on several definitions: (i) those
who improve by at least 2 points based on IGA-FWS versus Baseline;
(ii) those who improve by at least 1 point based on IGA-FWS versus
Baseline; (iii) those who have IGA-FWS scores of 0 or 1; (iv) those
who improve by at least 1 point based on PFWS; (v) those who
improve by at least 2 points based on PFWS; (vi) those who have a
score of at least 1 on GAIS scale;
[0132] (2) Secondary endpoints based on various definitions of
duration of response. Subjects who did not achieve an improvement
as specified in each definition below by Week 4 were assigned 0
duration; subjects who achieved an improvement as defined below but
did not revert back to baseline by Week 36 were censored at Week 36
(date of last evaluation). Treatment groups were compared using the
log rank test. For each definition and treatment group, a risk to
benefit ratio was computed as described above. Definitions for
duration of response include (i) time from injection to GAIS score
less than 1 for a responder definition of at least 1 in GAIS; (ii)
time from injection to reversion to baseline for a responder
definition of at least 2 point improvement in IGA-FWS; (iii) time
from injection to reversion to baseline for a responder definition
of at least 1 point improvement in PFWS; (iv) time from injection
to reversion to baseline for a responder definition of at least 2
point improvement in PFWS; (v) time from injection to reversion to
baseline for a responder definition of at least 1 point improvement
in IGA-FWS using proportional hazards model with term for
treatment, baseline severity, and treatment by baseline severity
interaction; and (vi) time from injection to reversion to baseline
for a responder definition of at least 1 point improvement in PFWS
using proportional hazards model with term for treatment, baseline
severity, and treatment by baseline severity interaction. An
exploratory analysis was conducted to correlate the subject's GAIS
assessment scores with the responder rates based on the PFWS for
both 1- and 2-point changes. Correlation analyses and logistic
regressions were used, as appropriate.
Interim Analysis and Study Results for the Treatment of Glabellar
Lines
[0133] An interim analysis of the primary variable was conducted
when 100% of the subjects had completed (or had previously
discontinued) the Week 12 visit. The analysis provided point
estimates with 95 percent confidence intervals for the proportion
of responders as defined in the primary endpoint. A point estimate
with a 95 percent confidence interval was also provided for the
difference in the proportion of responders between the active
comparator (Treatment Group 5) and each active treatment group
RT002 20 U, RT002 40 U and RT002 60 U (Treatment Groups 1, 2 and
3). Interim analyses of the secondary efficacy endpoints were
conducted for the Week 12 and the Week 24 visit when 75% of
subjects had completed these visits, respectively. The interim
analysis conducted at week 24 for safety and efficacy included at
least 75% (actual 100%) of subjects who had completed, or
previously discontinued, the week 24 visit. The analysis also
included primary and secondary efficacy endpoints (at maximum
frown) and safety data. As of the time of the interim analysis,
staff and subjects remained blinded. The results are summarized
hereinbelow.
[0134] Efficacy: RT002 achieved 100% response rates in all dose
groups at the 28-day primary efficacy endpoint of a 1-point
improvement on the IGA-FWS. (Placeholder for patient success).
RT002 doses maintained a 1-point benefit longer than the BOTOX.RTM.
Cosmetic. RT002 performed in a manner that was superior to
BOTOX.RTM. Cosmetic as measured by percentage of patients with none
or mild wrinkles. More than twice as many subjects treated with
RT002 40 U and 60 U maintained none or mild wrinkles (as per the
IGA-FWS scoring system) as compared to treatment with BOTOX.RTM.
Cosmetic at Week 16 (P<0.05) The percentage of subjects with
none or mild wrinkles (as per the IGA-FWS scoring system) was
statistically superior in all three RT002 dose groups compared to
BOTOX.RTM. Cosmetic at Week 16. The percentage of subjects with
none or mild wrinkles (as per the IGA-FWS scoring system) was
statistically superior in the RT002 40 U dose group compared to the
BOTOX.RTM. Cosmetic treatment group at all timepoints post Week
16.
[0135] Patient Data:
[0136] Patient reported outcomes supported investigator findings of
duration and efficacy of RT002 treatment. At 24 Weeks (6 months),
the 40 U RT002 dose continued to deliver clinically meaningful
higher response rates on the Subject Global Aesthetic Improvement
Scale (GAIS) with 46.3% of the RT002 40 U-treated subjects versus
31% of BOTOX.RTM. Cosmetic-treated subjects having a rating score
of at least a 1. At week 16, compared with the "up to 120 days"
duration of BOTOX.RTM. Cosmetic, based on its label information,
RT002 40 U dose achieved statistically significant higher response
rates as measured by at least a 1-point improvement on the Patient
Wrinkle Severity (PWS) Scale and at least a 1-point rating on the
Subject Global Aesthetic Improvement Scale. 76.9% of subjects
treated with RT002 40 U maintained at least a 1-point improvement
on PWS compared with 58.5% of subjects treated with BOTOX.RTM.
Cosmetic. In addition, 89.7% of subjects treated with RT002 40 U
maintained at least a 1-point score on GAIS compared with 70.7% of
subjects treated with BOTOX.RTM. Cosmetic.
[0137] Safety:
[0138] The RT002 product exhibited a safety and efficacy profile
highly comparable to BOTOX.RTM. Cosmetic. Adverse events were
generally mild, and were mainly associated with effects from the
injection itself. All RT002 dose groups exhibited an excellent
overall safety profile with AEs that were predominantly localized,
transient and mild in severity. No serious AEs occurred in any
active dose group. The 20 U and 40 U RT002 dose groups were well
tolerated and clinically superior to BOTOX.RTM. with respect to
causing Ptosis. In addition, RT002 exhibited less downward spread
at the 20 U an 40 U doses. Both the 20 U and the 40 U doses cause
No Ptosis in any subject treated with those doses of RT002 at any
time point, compared to 1.9% in the BOTOX.RTM. Cosmetic treated
group. A 5.7% ptosis rate was observed in subjects of the RT002 60
U treatment group. These were transient in nature, as typically
seen with BOTOX.RTM. treatment. The reduced diffusion of RT002 is
consistent with nonclinical and prior studies and supports a
reduced spread of toxin, as observed in subjects treated with
compositions of the invention which contain botulinum toxin, such
as botulinum toxin A, and a positively charged carrier comprising a
backbone, such as polylysine, with one or more covalently attached,
positively charged efficiency groups as described herein, such as
RT002.
[0139] Dosage and Duration of Effect:
[0140] Without wishing to be limiting, the interim analysis results
support a dose selection of 40 U as an optimal dose for single
treatment with the botulinum containing compositions of the
invention, based on the high responder rates, duration of effect
and positive safety profile. In addition, the compositions of the
invention, such as RT002, have a sustained and long lasting
duration of effect, e.g., for at least 6 months, following
administration by injection to a subject. As determined from the
interim analysis of the study results, treatment of subjects'
glabellar lines with the RT002 product achieved a superior duration
effect when compared to treatment of glabellar lines in subjects
with the BOTOX.RTM. Cosmetic. Indeed, a 5.9-month median duration
of 1-point improvement on IGA in the RT002 40 U dose group (23.6
weeks) was demonstrated versus an 18.8 week duration in subjects
treated with BOTOX.RTM. Cosmetic (p=0.020) based on Kaplan Meier
analysis method. (See, e.g., FIGS. 4A and 4B). Of note, at month 6,
a significant number of RT002-treated subjects were censored from
the interim analysis of duration since they were still responders.
At month 6, nearly one third (.about.33%) of the subjects in the
RT002 40 U treatment group still had no, or almost no, wrinkles
after a single treatment (p=0.041) versus 12% of subjects in the
BOTOX.RTM. Cosmetic treatment group. Further, the high dose group
was followed for 32 weeks post-treatment to assess duration of
response and achieved a median duration of 29.4 weeks or 7.3 months
based on both investigator and subject assessments.
[0141] The duration of effect provided by compositions of the
invention, such as RT002, as well as treatment methods and uses
thereof afford advantages in that subjects undergoing treatment
consider that duration of effect following treatment is of high
importance to them for an aesthetic treatment. Such a long,
sustained duration of effect, particularly achieved by a single or
one-time injection dose of product, namely, RT002, permits fewer
injections per treatment course for a subject, which is important
for the subject's comfort, convenience and overall well-being. A
product that affords significant and sustained effects, which are
maintained for at least a 6-month period following a single
treatment dose by injection of the product to a subject provides a
solution to an unmet need in the art for both practitioners and
patients.
[0142] Summary of Interim Results:
[0143] The results demonstrate that a composition of the invention
as represented by the RT002 product proved superior to BOTOX.RTM.
Cosmetic as measured by median duration of effect and responder
rates at 1-point and 2-point improvement on IGA-FWS, and percentage
of patients who achieved and maintained no wrinkles or mild
wrinkles pursuant to the IGA-FWS scoring system described above.
The study achieved statistically significant results for the
primary efficacy endpoint of 1-point improvement on IGA-FWS at 28
days. The week 24 interim analysis demonstrated clinically
meaningful differentiation in results afforded by single treatment
of subjects with an injected dose of RT002 versus injection with
BOTOX.RTM. Cosmetic.
[0144] As also determined by the interim analysis, RT002 achieved
an approximately 6-month duration of effect with high responder
rates. RT002 achieved superior duration of effect compared with
BOTOX.RTM. Cosmetic, demonstrating a 5.9-month median duration of
1-point improvement in glabellar lines based on the Investigator
Global Assessment-Facial Wrinkle Severity (IGA-FWS) scale in the 40
U dose group (23.6 weeks) versus 18.8 weeks for BOTOX.RTM. Cosmetic
(p=0.020) based on Kaplan Meier analysis method. At 24 weeks (6
months), RT002 at doses of 40 U and 60 U continued to deliver
clinically meaningful higher response rates with 35.9% and 29.3% of
subjects, respectively, maintaining a 1-point improvement versus
19% of BOTOX.RTM. Cosmetic-treated subjects. RT002 achieved its
primary efficacy endpoint of at least 1-point improvement on the
investigator scale (IGA-FWS) at 28 days, as well as the patient
reported outcome. RT002 achieved 100% response rates in all dose
groups at the 28-day primary efficacy endpoint of a 1-point
improvement on the Investigator Global Assessment Facial Wrinkle
Severity Scale (IGA-FWS). RT002 achieved greater than 97% response
rates in all dose treatment groups at the 28-day primary efficacy
endpoint of a 1-point improvement on the Patient Facial Wrinkle
Scale. Efficacy data showed 96% of subjects were rated with None or
Mild wrinkle severity at maximum frown 4 weeks post-treatment by
the clinical investigator assessment and 83% of subjects assessed
themselves as achieving None or Mild wrinkles at maxium frown at
the same time point. RT002 was well tolerated and no serious
adverse events were found. No eyelid Ptosis occurred in subjects in
the RT002 20 U or 40 U dose treatment groups. Dose response was
observed in the study; subjects who were administered the 40 U dose
of RT002 showed particularly high response rates.
[0145] Overall, DaxibotulinumtoxinA for Injection has been well
tolerated at all dose levels without any systemic or local safety
concerns or evidence of spread. RTT150 for Injection has been well
tolerated in clinical trials with no evidence of spread beyond the
treatment site at any dose. Adverse events in the phase 1/2
dose-escalating, open label clinical trial, RT002-CL001, were
generally mild, localized and transient. The most common adverse
events observed were headache and injection site reactions. No
subject in any cohort experienced ptosis. There were no serious
adverse events and adverse event rates did not change in frequency,
severity, or type with increasing doses. Thirty-four (34) subjects
reported 131 AEs. The most common adverse events reported were
headache (31 reports; 17 subjects); injection site pruritus (34
events; 8 subjects), injection site pain (burning) (14 events; 6
subjects), and eye disorders (14 events; 5 subjects). In addition
to adverse events, safety evaluations in the RT002-CL001 study
included clinical laboratory tests (hematology, chemistry,
urinalysis, and prothrombin time), serum antibodies for RTT150
toxin and RTP004 peptide, assessment of cranial nerves II-VII and
facial muscle strength, concomitant therapy medication and urine
pregnancy test for women of childbearing potential. There was no
evidence of spread beyond the treatment site at any dose and no
evidence of any systemic exposure based on clinical laboratory
results and physical assessments. All subjects were negative for
antibodies to both toxin and peptide.
Example 6
Follow Up Study Regarding Injectable Botulinum Toxin Formulation
Showing Long-Lasting Duration Effects in the Treatment of Glabellar
Lines
[0146] RT002 also was evaluated in a phase 2, dose-ranging, active
and placebo controlled clinical trial, RT002-CL002, in Canada, to
evaluate the safety, efficacy and duration of a single
administration for the treatment of moderate to severe glabellar
lines in adults. The trial enrolled 268 subjects (over 50 per
treatment group), who were treated with 20, 40 or 60 U of RT002, 20
U of BOTOX Cosmetic, or placebo. For the treatment of glabellar
lines, the proposed dosing regimen in the clinical trial was a
single treatment of 20, 40 or 60 Units per subject, 0.1 mL
intramuscular injection into each of 5 injections sites on the
forehead. Doses of 16, 32, 48, or 64 U based on current saline
potency method (corresponding to 25, 50, 75 and 100 U in previous
gelatin phosphate buffer potency method) were well tolerated in a
phase 1/2 clinical trial (Study RT002-CL001; 12 subjects per dose
group; 48 subjects total).
[0147] The interim data showed that RT002 achieved its primary
efficacy measurement for all three doses at 4 weeks. The study
demonstrated 6-month RT002 median duration of effect based upon at
least 1-point improvement in glabellar lines at maximum frown on
the Investigator Global Assessment-Facial Wrinkle Severity scale.
Subject-reported outcomes were consistent with investigator
findings of duration and efficacy of RT002. Across all cohorts,
RT002 appeared to be generally safe and well-tolerated. Adverse
events were generally mild, localized and transient. There were no
serious adverse events or evidence of any systemic exposure at any
of the three doses evaluated.
Sequence CWU 1
1
10151PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(20)This region may encompass
0-20 'Gly' residues wherein some positions may be
absentMISC_FEATURE(32)..(51)This region may encompass 0-20 'Gly'
residues wherein some positions may be absentSee specification as
filed for detailed description of substitutions and preferred
embodiments 1Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Arg Gly Arg Asp Asp Arg Arg
Gln Arg Arg Arg Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Gly 50
251PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(20)This region may encompass
0-20 'Gly' residues wherein some positions may be
absentMISC_FEATURE(32)..(51)This region may encompass 0-20 'Gly'
residues wherein some positions may be absentSee specification as
filed for detailed description of substitutions and preferred
embodiments 2Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Gly 50
349PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptideMISC_FEATURE(1)..(20)This region may encompass
0-20 'Gly' residues wherein some positions may be
absentMISC_FEATURE(30)..(49)This region may encompass 0-20 'Gly'
residues wherein some positions may be absentSee specification as
filed for detailed description of substitutions and preferred
embodiments 3Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Gly Gly 20 25 30 Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly 435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Lys Lys Lys Lys Lys Lys 1
5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly Arg Lys Lys Arg Arg
Gln 20 25 30 Arg Arg Arg 35 545PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptideMISC_FEATURE(1)..(20)This
region may encompass 0-20 'Gly' residues wherein some positions may
be absentMISC_FEATURE(21)..(45)This region may encompass 5, 7, 9,
11, 13, 15, 17, 21, 23 or 25 'Arg' residues wherein some positions
may be absentSee specification as filed for detailed description of
substitutions and preferred embodiments 5Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30 Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45
619PRTDrosophila sp. 6Ser Gly Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp 1 5 10 15 Lys Lys Cys 739PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
7Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Lys Lys Lys Lys 1
5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly Tyr Gly Arg
Lys 20 25 30 Lys Arg Arg Gln Arg Arg Arg 35 839PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Arg Gly Arg Asp Asp Arg Arg Gln Arg Arg Arg Gly Lys Lys Lys Lys 1
5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Gly Arg Gly Arg
Asp 20 25 30 Asp Arg Arg Gln Arg Arg Arg 35 920PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMISC_FEATURE(1)..(20)This sequence may encompass 10-20 'Lys'
residues wherein some positions may be absentSee specification as
filed for detailed description of substitutions and preferred
embodiments 9Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys 20 1010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Gly
Gly Gly Arg Arg Arg Arg Arg Arg Arg 1 5 10
* * * * *