U.S. patent application number 16/614773 was filed with the patent office on 2020-06-11 for methods of treatment for cervical dystonia.
The applicant listed for this patent is Revance Therapeutics, Inc.. Invention is credited to Curtis L. Ruegg, Jacob M. Waugh.
Application Number | 20200179498 16/614773 |
Document ID | / |
Family ID | 64274745 |
Filed Date | 2020-06-11 |
United States Patent
Application |
20200179498 |
Kind Code |
A1 |
Ruegg; Curtis L. ; et
al. |
June 11, 2020 |
METHODS OF TREATMENT FOR CERVICAL DYSTONIA
Abstract
This invention provides methods to treat or prevent cervical
dystonia, a disorder related thereto, or a symptom thereof, with
novel injectable compositions comprising botulinum toxin that may
be administered to a subject suffering from such maledy. The
injectable compositions and methods in which these compositions are
used provide novel and advantageous treatments which result in high
responder rates and long duration of effect, for example, a
duration of effect for 24 weeks and longer.
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: |
64274745 |
Appl. No.: |
16/614773 |
Filed: |
May 18, 2018 |
PCT Filed: |
May 18, 2018 |
PCT NO: |
PCT/US18/33397 |
371 Date: |
November 18, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62508324 |
May 18, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 21/00 20180101;
C07K 14/33 20130101; A61K 9/0019 20130101; A61K 38/4893 20130101;
A61K 47/42 20130101; A61K 47/26 20130101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 47/26 20060101 A61K047/26; A61K 47/42 20060101
A61K047/42; A61P 21/00 20060101 A61P021/00 |
Claims
1. A method of administering botulinum toxin to achieve an extended
duration therapeutic effect in an individual with cervical
dystonia, the method comprising: administering by injection a first
treatment dose of a sterile injectable composition into one or more
of the muscles causing the cervical dystonia in the individual in
need of treatment to achieve the therapeutic 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(SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), wherein the
subscripts p and q are each independently an integer of from 0 to
20; wherein the total treatment dose of botulinum toxin component
administered to the individual is 100 U to 450 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 24 week duration of effect, optionally,
before a second or subsequent treatment dose is administered.
2. A method of treating cervical dystonia in an individual in need
thereof, the method comprising: administering to the individual by
injection to one or more muscles causing the cervical dystonia in
the individual a composition comprising: a pharmaceutically
acceptable diluent for injection; a botulinum toxin components
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(SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 a total treatment dose amount of 100 U to 450 U; wherein the
positively charged carrier is non-covalently associated with the
botulinum component; and wherein the injection of the composition
provides a single treatment dose having at least about a 24 week
duration of effect in reducing the symptoms of cervical dystonia 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 effect in an individual with cervical
dystonia, said composition comprising a pharmaceutically acceptable
diluent suitable for injection; a botulinum toxin component in a
treatment dose of 100 U to 450 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(SEQ ID
NO: 1), (gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 effect having at least about a 24 week duration of
effect in the individual administered said formulation by
injection.
4. A pharmaceutical composition in a sterile injectable formulation
for use in reducing the symptoms of cervical dystonia in an
individual in need thereof, said composition comprising: a
botulinum toxin component in a dose of 100 U to 450 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(SEQ ID
NO: 1), (gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 24
week duration of effect in reducing the symptoms of cervical
dystonia 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 achieves the extended duration therapeutic
effect for at least about a 8 months.
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 (SEQ ID NO: 1), 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 (SEQ ID NO: 2), 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 (SEQ ID NO: 3), 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 (SEQ ID NO: 4).
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 treatment dose of botulinum
toxin is administered to the individual in an amount of about 100 U
to 200 U.
16. The method or pharmaceutical composition for use according to
any one of claims 1 to 14, wherein the treatment dose of botulinum
toxin is administered to the individual in an amount of about 200 U
to 300 U.
17. The method or pharmaceutical composition for use according to
any one of claims 1 to 14, wherein the treatment dose of botulinum
toxin is administered to the individual in an amount of 300 U to
450 U.
18. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 3 months.
19. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 4 months.
20. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 5 months.
21. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 6 months.
22. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 7 months.
23. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 8 months.
24. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises greater than 9 months.
25. The method or pharmaceutical composition for use according to
any one of claims 1 to 17, wherein the duration of treatment effect
comprises at least 6 months through 10 months.
26. 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 100 U, 200 U, 300 U or 450 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 (SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 24 weeks following a single treatment of an individual with
the injectable composition.
27. The composition according to claim 26, wherein the positively
charged carrier has the amino acid sequence
RKKRRQRRRG-(K).sub.15-GRKKRRQRRR (SEQ ID NO: 4).
28. The composition according to claim 26 or claim 27, wherein the
composition comprises botulinum toxin of serotype A.
29. The composition according to claim 28, wherein the composition
comprises botulinum toxin of serotype A having a molecular weight
of 150 kDa.
30. The composition according to any one of claims 26 to 29,
wherein the treatment dose of botulinum toxin is administered to
the individual in the amount of 100 U.
31. The composition according to any one of claims 26 to 29,
wherein the treatment dose of botulinum toxin is administered to
the individual in the amount of 200 U.
32. The composition according to any one of claims 28 to 31,
wherein the treatment dose of botulinum toxin is administered to
the individual in the amount of 450 U.
33. A method of treating an individual with cervical dystonia 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 one or more muscles causing
cervical dystonia in the individual in need of treatment to achieve
a therapeutic effect of reducing the symptoms of cervical dystonia
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(SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 about 100 U to about 450 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 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 6
months to at least about 10 months following the initial treatment
dose and between each subsequent treatment dose.
34. The method according to any one of claim 33 to claim 35,
wherein the composition comprises botulinum toxin of serotype
A.
35. The method according to claim 34, wherein the composition
comprises botulinum toxin of serotype A having a molecular weight
of 150 kDa.
36. The method according to any one of claims 33 to 35, 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 (SEQ ID NO:
1), wherein the subscripts p and q are each independently an
integer of from 0 to 20.
37. The method according to any one of claims 33 to 35, 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 (SEQ ID NO:
2), wherein the subscripts p and q are each independently an
integer of from 0 to 20.
38. The method according to any one of claim 33 to 35, 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 (SEQ ID NO:
3), wherein the subscripts p and q are each independently an
integer of from 0 to 20.
39. The method according to any one of claims 33 to 38, 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.
40. The method according to any one of claims 33 to 39, 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.
41. The method according to any one of claims 33 to 35, wherein the
positively charged carrier has the amino acid sequence
RKKRRQRRRG-(K).sub.15-GRKKRRQRRR (SEQ ID NO: 4).
42. The method according to any one of claims 33 to 41, wherein the
composition does not locally diffuse from the site of injection
following injection.
43. The method according to any one of claims 33 to 42, wherein the
botulinum toxin is administered to the individual in an amount of
about 100 U to 200 U.
44. The method according to any one of claims 33 to 42, wherein the
botulinum toxin is administered to the individual in an amount of
200 U to 300 U.
45. The method according to any one of claims 33 to 42, wherein the
botulinum toxin is administered to the individual in an amount of
300 U to 450 U.
46. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 3
months.
47. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 4
months.
48. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 5
months.
49. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 6
months.
50. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 7
months.
51. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 8
months.
52. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises greater than 9
months.
53. The method according to any one of claims 33 to 45, wherein the
duration of the treatment interval comprises at least 6 months
through 10 months.
Description
SEQUENCE LISTING
[0001] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 13, 2016, is named CD.txt and is 6,809 bytes in size.
FIELD OF THE INVENTION
[0002] This invention relates to methods to treat or prevent
cervical dystonia, a disorder related thereto, or a symptom
thereof, with novel injectable compositions comprising botulinum
toxin that may be administered to a subject suffering from such
maledy. The injectable compositions and methods in which these
compositions are used provide novel and advantageous treatments
which result in high responder rates and long duration of effect,
for example, a duration of effect for over 24 weeks.
BACKGROUND OF THE INVENTION
[0003] Cervical Dystonia is an extremely painful, chronic
neurological movement disorder where the neck and shoulder muscles
contract involuntarily and contort, causing causing abnormal
movements and awkward posture of the head and neck such as the head
to twist or turn to the left or right (torticollis), upwards
(retrocollis), downwards (antecollis) or sideways (laterocollis).
The movements may be sustained (tonic), jerky (clonic), or a
combination. Cervical dystonia (also referred to as Neck Dystonia
or Spasmodic Torticollis) affects a person's ability to control
muscle activity. Cervical dystonia may be primary (meaning that it
is the only apparent neurological disorder, with or without a
family history) or may be brought about by secondary causes (such
as physical trauma) and is often attributed to nervous system
damage caused by a stroke, disease or trauma. A rare disorder that
can occur at any age, even during infancy, cervical dystonia most
often occurs in middle-aged individuals, and is more prevalent in
women than men. Those with a family history of cervical dystonia or
some other type of dystonia are at higher risk of developing the
disorder.
[0004] Cervical dystonia is the third most common movement disorder
following essential tremor and Parkinson's disease. An estimated 3
in every 10,000 people are known to suffer from cervical dystonia.
The number of cases reported in North America alone is
approximately 300,000.
[0005] Symptoms generally begin gradually and then reach a plateau
where the symptoms don't get substantially worse. Unfortunately,
there is no cure for cervical dystonia and the condition greatly
impacts an individual's quality of life. In some cases, the
disorder resolves without treatment, but sustained remissions are
fairly uncommon.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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 therapeutic purposes to
treat cervical dystonia.
[0013] Treatments for cervical dystonia include oral medications,
botulinum toxin injections, surgery, and complementary therapies.
The most commonly prescribed treatment for cervical dystonia is the
use of botulinum toxin, typically type A (although Type B has also
been used), which can reduce its signs and symptoms. Botulinum
toxin can help block the communication between the nerve and the
muscle and may alleviate abnormal movements and postures. The
number of injections is typically based on the severity of the
dystonia. Doctors injecting the toxin may select the muscles to be
injected by observing abnormal postures or movements and feeling
for the muscle spasm or by using an electromyography machine to
measure muscle activity. Each muscle affected by dystonia typically
has to be injected separately. As such, based on the diffusion
characteristics of currently available toxin formulations, there is
a limit to the total quantity of toxin that can be injected into
the body at one time. While the treatment for cervical dystonia
involves regular neurological intervention, which takes effect over
a period of 4-7 days or longer after injection, the response to the
treatment with botulinum toxin typically wears off after a 12 week
period, often as early as at 10 weeks, requiring the person
suffering from cervical dystonia to be injected again. Therefore, a
durable, longer acting treatment requiring fewer neurological
interventions would be desirable.
SUMMARY OF THE INVENTION
[0014] In one of its aspects, the invention relates to a method for
producing a biologic effect in the treatment of cervical dystonia
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.
[0015] In another of its aspects, this invention provides a method
of treating cervical dystonia in an individual in need thereof, the
method comprising administering to the individual an injection of 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 (SEQ ID NO: 1), (gly)p-YGRKKRRQRRR-(gly)q
(SEQ ID NO: 2) or (gly)p-RKKRRQRRR-(gly)q (SEQ ID NO: 3), 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 from about 100 U to about 450 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 injection of the composition provides a
treatment having at least about a six month to about a 10 month
duration of effect in reducing the symptoms of cervical dystonia,
thereby extending treatment interval duration for the
individual.
[0016] In another of its aspects, this invention provides
injectable compositions comprising botulinum toxin non-covalently
associated with a positively charged carrier molecule used to treat
cervical dystonia. 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.
[0017] A further 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 in the
treatment of cervical dystonia. 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.
[0018] 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 for use
in a method of treatment for cervical dystonia.
[0019] In another aspect, the invention provides effective doses
and amounts of the compositions of this invention in the treatment
of cervical dystonia 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 effective doses and
amounts that produce or result in a desired therapeutic effect in a
subject to whom the doses and amounts are administered. In
particular embodiments, 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 dose amounts of
about 100 U to about 450 U per subject, afforded a response rate of
in the reduction of cervical dystonia symptoms for at least 16
weeks, at least 20 weeks, at least 24 weeks, or about 6 to 10
months, or even longer. 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 as well as by the described treatment
methods and uses, affords significant advantages compared to the
art. By way of example, subjects undergoing 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 and treatment regime 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 16 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 effect in an individual suffering from cervical
dystonia, 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 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 (SEQ ID NO: 1),
(gly)p-YGRKKRRQRRR-(gly)q (SEQ ID NO: 2) or (gly)p-RKKRRQRRR-(gly)q
(SEQ ID NO: 3), 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
about 100 U to 450 U; or more specifically, from about 100 U to 200
U or from about 200 U to 300 U or from about 300 U to 450 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 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 for the treatment
of cervical dystonia, a sterile injectable composition comprising a
botulinum toxin, a botulinum toxin complex, or a reduced botulinum
toxin complex in a dosage amount selected from more than 100 U,
from 100 U to 200 U, 200-300 U, or 300-450 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 (SEQ ID NO: 1), (gly)p-YGRKKRRQRRR-(gly)q
(SEQ ID NO: 2) or (gly)p-RKKRRQRRR-(gly)q (SEQ ID NO: 3), 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 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.
[0023] 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 (SEQ ID NO: 4). In an
embodiment, the botulinum toxin is present in the composition in a
dosage amount from more than 100 U, 100-200 U, or 200-300 U or
300-450 U. In an embodiment, the botulinum toxin is present in the
composition in a dosage amount selected from the group consisting
of 100 U, 200 U, 300 U and 450 U. In an embodiment, the composition
reduces the symptoms of cervical dystonia 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.
[0024] In another of its aspects, the invention provides a method
of treating an individual suffering from cervical dystonia 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 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 (SEQ ID NO: 1), (gly)p-YGRKKRRQRRR-(gly)q
(SEQ ID NO: 2) or (gly)p-RKKRRQRRR-(gly)q (SEQ ID NO: 3), 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 from more than 100 U, 100-200 U,
200-300 U, or 300-450 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 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.
[0025] In embodiments of the above-described treatment method, the
therapeutic effect is treatment of the symptoms of cervical
dystonia. 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 (SEQ ID NO:
[0026] 4). 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 more than 100 U, 100-200 U, 200-300 U, or 300-450
U. 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.
DESCRIPTION OF THE FIGURES
[0027] FIG. 1 shows the Cervical Dystonia Phase 2 Study Design as
described in the Example herein.
[0028] FIG. 2 shows the Demographics by Cohort in the Study as
described in the Example herein.
[0029] FIG. 3 shows Number (%) of Subjects with Treatment-Related
AE's by Cohort By Preferred Term in the Study as described in the
Example herein.
[0030] FIG. 4 shows Primary Endpoint by Cohort--Reduction in
TWSTRS-Total Score at Week 4 in the Study as described in the
Example herein.
[0031] FIG. 5 shows Secondary Endpoint by Cohort--Change from
Baseline in TWSTRS-Total Score over Time in the Study as described
in the Example herein.
[0032] FIG. 6 shows Secondary Endpoint by Cohort--Duration of
Response Defined by Subject Reaching Target-TWSTRS Score (of
subjects with improvement at Week 4. Withdrawals due to need for
retreatment are considered as events) in the Study as described in
the Example herein.
[0033] FIG. 7 shows Subject Distribution by Dose--Study Doses
administered cluster into Two Separate Dose Groups in the Study as
described in the Example herein.
[0034] FIG. 8 shows Secondary Endpoint by Dose Group--Change from
Baseline in TWSTRS-Total Score over Time as described in the
Example herein.
DETAILED DESCRIPTION OF THE INVENTION
[0035] This invention relates to novel injectable compositions
comprising botulinum toxin, a botulinum toxin complex, or a reduced
botulinum toxin complex used in method to treat cervical dystonia.
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, 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 100 U
or more, are administered by injection for the treatment of
cervical dystonia.
[0036] 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..
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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. 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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).sub.n2 (SEQ ID NO: 5), 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 (SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3) 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 (SEQ ID
NO: 6). In other embodiments, the positively charged carrier is a
positively charged peptide having the amino acid sequence
RKKRRQRRR-G-(K).sub.15-G-RKKRRQRRR (SEQ ID NO: 4); or a positively
charged peptide having the amino acid sequence
YGRKKRRQRRR-G-(K).sub.15-G-YGRKKRRQRRR (SEQ ID NO: 7); or a
positively charged peptide having the amino acid sequences
RGRDDRRQRRR-G-(K).sub.15-G-RGRDDRRQRRR (SEQ ID NO: 8) for use in
the compositions and methods of the invention.
[0050] 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 (SEQ ID NO:
5), a preferred range is from about 0.1 to about 25%.
[0051] 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 (SEQ ID NO: 9), 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 (SEQ
ID NO: 10)) or HIV-TAT.
[0052] 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.
[0053] 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.
[0054] 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.).
[0055] 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.
[0056] 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 (LD50 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.
[0057] Compositions of this invention are preferably in a form that
permits injection into the skin or epithelium of subjects or
patients. The term "in need" is meant to include both
pharmaceutical or health-related needs (e.g., treating conditions
involving undesirable dystonic contractions or muscle spasms). 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
"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.
[0058] 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.
[0059] 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).
[0060] 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.
[0061] 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).
[0062] 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%.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] The compositions of the invention are administered to
deliver an effective amount, preferably a therapeutically effective
amount, of the botulinum toxin. The term "effective amount" or
"therapeutically 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 effect, but that
implicitly is a safe amount, i.e., one that is low enough to avoid
serious side effects.
[0067] 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 and/or sequential applications over periods of time.
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 cervical dystonia. The botulinum toxin
is administered by injection to muscles or to other skin-associated
or other target tissue structures.
[0068] 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. In the treatment of cervical dystonia, the following Table
1 provides guidance as to the appropriate dosage of RTT150 (the
RT002 product is composed of purified 150 kDa botulinum neurotoxin,
referred to as RTT150) by dose ranges 1, 2 and 3 for specified
muscle groups:
[Table 1 was created using the perspective of the recommended
dosing ranges for BOTOX.RTM. by involved muscle (Allergan, Inc.
BOTOX.RTM. US Prescribing Information, 2015 & BOTOX.RTM.
medical website, Flexible dosing in cervical dystonia.
https://www.botoxmedical.com/CervicalDystonia/DosingAndAdministration)]
TABLE-US-00001 TABLE 1 General Guideline on Dosing Ranges by Muscle
for Cervical Dystonia, RTT150 for Injection - Dose Range 1 Dose
Range 2 Dose Range 3 Total maximum Per subject: Per subject: 200
Per subject: 300 dose up to 200 U to 300 U to 450 U RTT150 RTT150
RTT150 By muscle Recommended Recommended Recommended dose range
dose range dose range RTT150 Units RTT150 Units RTT150 Units
Splenius capitis 15-100a 15-100 22.5-150 Sterno- 15-100a 15-100a
22.5-100a cleidomastoid Levator scapulae 20-100a 20-100a 30-150
Scalene complex 15-50a 15-50a 22.5-75 Semispinalis 30-100a 30-100a
45-150 capitis Longissimus 30-100a 30-100a 45-150 Trapezius 20-100a
20-100a 30-150 Splenius cervicis 20-60a 20-60a 30-90
[0069] 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. 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, or series of injections
during a single multiple 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
about 100 U to 450 U; or more specifically, from about 100 U to 200
U or from about 200 U to 300 U or from about 300 U to 450 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.
[0070] 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 about 100 U to 450 U; or more specifically, from about 100
U to 200 U or from about 200 U to 300 U or from about 300 U to 450
U, of botulinum toxin per treatment dose per subject for the
treatment of cervical dystonia. According to the invention, a
treatment effectendures for several weeks or months, for example,
for at least 10 weeks, for at least 12 weeks, for at least 16
weeks, 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 (SEQ
ID NO: 5), 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 (SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 (SEQ ID NO: 4). 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 (SEQ ID NO: 1),
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q (SEQ ID NO: 2) or
(gly).sub.p-RKKRRQRRR-(gly).sub.q (SEQ ID NO: 3), 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 (SEQ ID NO: 4). 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 about 100 U to 450
U; or more specifically, from about 100 U to 200 U or from about
200 U to 300 U or from about 300 U to 450 U, of botulinum toxin and
a response or effect is achieved and maintained for a long
duration, e.g., for at least 10 weeks, for at least 12 weeks, for
at least 16 weeks, for 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.
[0071] 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
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
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 about 100 U to 450 U; or more
specifically, from about 100 U to 200 U or from about 200 U to 300
U or from about 300 U to 450 U, of botulinum toxin in the
composition.
[0072] 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).
[0073] 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.
[0074] 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.
[0075] 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.
[0076] In some embodiments, the patient to be treated 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.
EXAMPLES
Example 1
[0077] This example is an open-label, sequential, dose-escalating
clinical study of DaxibotulinumtoxinA Injectable (RT002) to treat
moderate-to-severe isolated cervical dystonia, a movement disorder
of the neck, in adults. Thirty-seven subjects with
moderate-to-severe cervical dystonia were enrolled at multiple
sites in the United States. The trial's first cohort of 12 subjects
received a single dose of up to 200 units of RT002 injectable, the
second cohort of 12 subjects received between 200 and 300 units,
and the third cohort of 13 subjects received from 300 to 450 units.
The study showed positive efficacy results and that RT002 was
generally safe and well-tolerated.
[0078] All subjects were followed until they returned to baseline
or for up to a total of 24 weeks after treatment. Due to the long
duration of effect seen in the first cohort, subjects in the second
and third cohorts were given the option to continue. Several
patients elected to remain in the study and will be followed for up
to 36 weeks.
[0079] The primary efficacy endpoint of the Phase 2 study was an
improvement in dystonia symptoms as measured by change (reduction)
from baseline in Toronto Western Spasmodic Torticollis Rating Scale
(TWSTRS)-Total score at four weeks. TWSTRS is a validated composite
scale that covers different features of the cervical dystonia
condition. The first part of the scale is based on the physical
findings and severity of dystonia, the second part rates the
patient's perceived level of disability, and the third part rates
pain associated with the condition. The study protocol also feature
a number of secondary efficacy endpoints.
[0080] In sum, the Study Objectives were: [0081] To assess the
safety and preliminary efficacy of RT002 for Injection in subjects
with isolated Cervical Dystonia and [0082] To evaluate the duration
of effect of RT002 for Injection in the treatment of isolated
Cervical Dystonia
[0083] The Primary Endpoint was: [0084] improvement of dystonia, as
measured by change from baseline in TWSTRS-Total score at Week 4
(TWSTRS=Toronto Western Spasmodic Torticollis Rating Scale).
[0085] The Secondary Endpoints were: [0086] Change from baseline in
TWSTRS-Total score; [0087] Change from baseline in TWSTRS subscale
scores: (i.e. TWSTRS-Severity Scale, TWSTRS-Disability Scale &
TWSTRS-Pain Scale); [0088] Duration of effect, as assessed by the
number of weeks from treatment until return of symptoms that
warrant treatment, regarded as when a subject reaches or exceeds
their target TWSTRS-Total score, or subject expresses a need for
treatment and investigator agrees that it is necessary; [0089]
Percentage of responders showing improvement on CGIC (Clinician
Global Impression of Change); and [0090] Patient-rated quality of
life, measured by change from baseline in CDIP-58 (Cervical
Dystonia Impact Profile-58) Total score (all post-treatment time
points).
[0091] Top-Line 24-Week Results:
[0092] Duration of Effect at Least 24 Weeks:
[0093] The median duration of effect was at least 24 weeks for each
of the three dose cohorts studied. Duration of effect was defined
as the number of weeks from treatment until the return of signs and
symptoms that warrant retreatment, based on subjects reaching their
target Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS)
score. For reference, current treatment of cervical dystonia calls
for injection of botulinum toxin approximately every 3 months (12
weeks), or 4 times per year.
[0094] Positive Efficacy Results:
[0095] The trial's 4-week primary efficacy measurement was the
improvement in signs and symptoms of cervical dystonia as
determined by reduction of the TWSTRS-Total score from baseline. At
Week 4, RT002 injectable showed a clinically significant mean
reduction of 38% from baseline across all three cohorts. This
reduction continued to increase to 50% at Week 6 for all subjects,
was 42% at Week 12 and was maintained at or above 30% through Week
24. For reference, placebo-controlled trials for botulinum toxin
type A products approved to treat cervical dystonia had a reduction
in the TWSTRS-Total score from baseline of 21% to 26% at Week 4 and
13% to 16% at Week 12.
[0096] On the key secondary endpoint, percentage of responders
showing improvement on Clinician Global Impression of Change
(CGIC), 97% of all subjects experienced an improvement in cervical
dystonia symptoms at Week 4.
[0097] Generally Safe and Well-Tolerated:
[0098] In all three cohorts, RT002 injectable appeared to be
generally safe and well-tolerated through Week 24. There were no
serious adverse events and no dose-dependent increase in adverse
events. The treatment-related adverse events were generally
transient and mild to moderate in severity, with one case of neck
pain reported as severe. The most common adverse events were
dysphagia, or difficulty in swallowing (14%), of which all cases
were mild in severity, injection site redness (8%), injection site
bruising (5%), injection site pain (5%), muscle tightness (5%) and
muscle weakness (5%). For reference, trials for botulinum toxin
type A products approved to treat cervical dystonia have adverse
events for dysphagia ranging from 13% to 39%.
[0099] Patients with cervical dystonia suffer from considerable
pain and debilitation, which dramatically impacts their quality of
life. Nearly all subjects in this study responded to treatment and
a majority were still responding to RT002 at 24 weeks. FIGS. 1-8
provide additional data regarding the study design and results.
[0100] The Safety Summary of the Study:
[0101] RT002 appeared to be generally safe and well tolerated
through Week 24 across all dose groups evaluated, with no increase
in Treatment-Emergent Adverse Event's (TEAE's) upon dose
escalation.
[0102] Total of 22 Treatment-Related TEAE's reported in 13 of 37
subjects (35%) [0103] Most frequently reported: dysphagia (14%),
injection site erythema (8%), injection site bruising (5%),
injection site pain (5%), muscle tightness (5%) and muscular
weakness (5%) [0104] All TEAE's were mild or moderate in severity
except for one case of neck pain reported as severe (Day 10 onset,
duration of 2 days)
[0105] No Serious Adverse Events were reported.
[0106] All treatment-related TEAE's of special interest resolved
with similar or lower incidence rates vs. prior BoNTA studies
(Trials of other BoNTA products approved to treat CD have dysphagia
rates ranging from 13-39%; Includes BOTOX, Dysport and Xeomin. Data
as reported in product prescribing information). [0107] Dysphagia:
14% ( 5/37; all mild); average duration 35 days. [0108] Muscular
Weakness: 5% ( 2/37; 1 mild, 1 moderate), both local [0109] Neck
pain: 3% ( 1/37; severe)
[0110] The Efficacy Summary of the Study:
[0111] Duration of Effect:
[0112] The median duration of effect, defined as subjects
maintaining at least 20% of the treatment benefit achieved at Week
4 (Target TWSTRS Score), was >24 Weeks for each of the 3 cohorts
studied [0113] When analyzed by Dose Groups A and B, the median
duration of effect was >24 weeks for both groups
[0114] Improvement in CD Signs and Symptoms:
[0115] A clinically significant reduction from baseline in the
TWSTRS Total Score of 38% was observed at Week 4 across all
subjects [0116] The improvement from baseline peaked at 50% at Week
6, with the majority of this treatment benefit maintained at
>30% through Week 24
[0117] Global Impression of Change Response Rate:
[0118] 97% of all subjects experienced improvement (Score >1) at
Week 4 in their cervical dystonia symptoms as assessed by Clinician
Global Impression of Change (CGIC)
[0119] In a preferred embodiment of this invention the 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 (SEQ ID NO: 4), and which
does not contain accessory proteins or animal-derived components of
the preferred method of this invention a single (one-time)
treatment by injection of RT002 for the treatment of cervical
dystonia is administered, optionally at different muscle locations
on a subjects neck. The preferred treatment dose is more than 100
U, 100-200 U, 200-300 U and 300-450 U, of RT002 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.
[0120] Dosing regimen and injection technique: The preferred dosing
regimen of RT002 of this invention is a single treatment of RT002
(dosed at up to 100 U, 100-200 U, 200-300 U or 300-450 U per
subject, as a 0.1 mL intramuscular injection into injections sites
on the neck of the subject undergoing treatment.
[0121] 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.
[0122] Dosage and Duration of Effect: Without wishing to be
limiting, the interim analysis results support a dose selection of
more than 100 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. 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. 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.
[0123] 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.
[0124] All publications, patents, and published patent applications
cited herein are hereby incorporated by reference in their
entireties for all purposes.
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 Gly1 5 10 15Gly Gly Gly Gly Arg Gly Arg Asp Asp Arg Arg Gln
Arg Arg Arg Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 35 40 45Gly Gly Gly 50251PRTArtificial
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 Gly1 5 10 15Gly Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 35 40 45Gly Gly Gly 50349PRTArtificial
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 Gly1 5 10 15Gly Gly Gly Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Gly Gly Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 35 40 45Gly435PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 4Arg Lys Lys Arg Arg
Gln Arg Arg Arg Gly Lys Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys
Lys Lys Lys Lys Lys Gly Arg Lys Lys Arg Arg Gln 20 25 30Arg Arg Arg
35545PRTArtificial 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 Gly1 5 10 15Gly Gly Gly Gly Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 20 25 30Arg Arg Arg Arg
Arg Arg Arg Arg Arg Arg Arg Arg Arg 35 40 45619PRTDrosophila sp.
6Ser Gly Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp1 5
10 15Lys Lys Cys739PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Gly Tyr Gly Arg Lys 20 25 30Lys Arg Arg Gln Arg Arg Arg
35839PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Arg Gly Arg Asp Asp Arg Arg Gln Arg Arg Arg
Gly Lys Lys Lys Lys1 5 10 15Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Gly Arg Gly Arg Asp 20 25 30Asp Arg Arg Gln Arg Arg Arg
35920PRTArtificial 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 Lys1 5 10 15Lys Lys Lys Lys 201010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Gly
Gly Gly Arg Arg Arg Arg Arg Arg Arg1 5 10
* * * * *
References