U.S. patent application number 14/065379 was filed with the patent office on 2014-05-01 for compositions and methods for safe treatment of rhinitis.
This patent application is currently assigned to Revance Therapeutics, Inc.. The applicant listed for this patent is Revance Therapeutics, Inc.. Invention is credited to Curtis L. Ruegg, Hongran Fan Stone, Jacob M. Waugh.
Application Number | 20140120077 14/065379 |
Document ID | / |
Family ID | 50545513 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120077 |
Kind Code |
A1 |
Ruegg; Curtis L. ; et
al. |
May 1, 2014 |
Compositions and Methods for Safe Treatment of Rhinitis
Abstract
Methods for treating rhinitis in a subject are provided herein.
The methods of the present invention comprise intranasal
administration of a topical composition comprising a purified
botulinum neurotoxin, a carrier and a viscosity modifier to one or
more inner surfaces of the nose. The methods disclosed herein
provide alternative methods for delivery of botulinum neurotoxin to
the nasal anatomy for the treatment of rhinitis.
Inventors: |
Ruegg; Curtis L.; (Redwood
City, CA) ; Stone; Hongran Fan; (Beijing, CN)
; Waugh; Jacob M.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Revance Therapeutics, Inc. |
Newark |
CA |
US |
|
|
Assignee: |
Revance Therapeutics, Inc.
Newark
CA
|
Family ID: |
50545513 |
Appl. No.: |
14/065379 |
Filed: |
October 28, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61719462 |
Oct 28, 2012 |
|
|
|
Current U.S.
Class: |
424/94.63 |
Current CPC
Class: |
A61K 9/0043 20130101;
A61M 31/00 20130101; A61K 47/10 20130101; A61K 47/42 20130101; A61K
38/4893 20130101; A61K 38/48 20130101; A61K 47/34 20130101; C12Y
304/24069 20130101 |
Class at
Publication: |
424/94.63 |
International
Class: |
A61K 38/48 20060101
A61K038/48 |
Claims
1. A method for treating rhinitis, the method comprising
intranasally administering a topical botulinum toxin composition to
a patient in need of treatment; wherein said topical botulinum
toxin composition comprises a purified botulinum neurotoxin; a
carrier molecule comprising a positively charged backbone with
positively charged efficiency groups attached thereto; and a
viscosity modifier.
2. The method according to claim 1, wherein the purified botulinum
neurotoxin is isolated from a botulinum serotype selected from the
group consisting of botulinum toxin type A, B, C1, D, E, F, and
G.
3. The method according to claim 1, wherein the purified botulinum
neurotoxin is isolated from botulinum toxin type A.
4. The method according to claim 1, wherein the positively charged
backbone comprises a polypeptide.
5. The method according to claim 4, wherein the polypeptide
comprises polylysine.
6. The method according to claim 1 or 4, where the positively
charged efficiency groups are selected from the group consisting of
-(gly).sub.n1-(arg).sub.n2, (gly)p-RGRDDRRQRRR-(gly)q,
(gly)p-YGRKKRRQRRR-(gly)q, (gly)p-RKKRRQRRR-(gly)q wherein the
subscripts p and q are each independently an integer of from 0 to
20, and wherein n1 is an integer of from 0 to 20 and the subscript
n2 is independently an odd integer of from about 5 to about 25.
7. The method according to claim 1, wherein the viscosity modifier
is selected from the group consisting of polyoxyethylene glycol
alkyl ethers, polyoxypropylene glycol alkyl ethers, glucoside alkyl
ethers, polyoxyethylene glycol octylphenol ethers, polyoxyethylene
glycol alkylphenol ethers, glycerol alkyl esters, polyoxyethylene
glycol sorbitan alkyl esters, sorbitan alkyl esters,
dodecyldimethylamine oxide, block copolymers of polyethylene glycol
and polypropylene glycol (polyoxamers) and combinations
thereof.
8. The method according to claim 7, wherein the viscosity modifier
agent is poloxamer 407.
9. The method according to claim 8, wherein the poloxamer 407 is
present in the botulinum toxin composition at a concentration of
10-25%.
10. The method according to claim 9, wherein the poloxamer 407 is
present in the botulinum toxin composition at a concentration of
15-20%.
11. The method according to claim 1, wherein the topical botulinum
toxin composition is administered to a patient's inferior
turbinates.
12. The method according claim 1, wherein said topical botulinum
toxin composition contains botulinum toxin at a concentration in
the range of 1,000 U/mL to 20,000 U/mL.
13. The method according to claim 1, wherein the concentration of
botulinum toxin in the topical botulinum toxin composition is 5,500
U/mL, 5,750 U/mL, 6,000 U/mL, 6,250 U/mL, 6,500 U/mL, 6,750 U/mL or
7,000 U/mL.
14. The method according to claim 1, wherein the topical botulinum
toxin is applied using an applicator.
15. The method according to claim 14, wherein the applicator is a
swab.
16. The method according to claim 1, wherein the rhinitis selected
from the group consisting of infectious rhinitis, vasomotor
rhinitis, allergic rhinitis, rhinitis medicamentosa, atrophic
rhinitis, rhinitis sicca, and polypous rhinitis.
17. The method according to claim 16, wherein the rhinitis is
vasomotor rhinitis.
18. The method according to claim 1, wherein the purified botulinum
neurotoxin is 10-fold to 90-fold less toxic with respect to oral
ingestion by test animals than botulinum toxin complexed with
native accessory proteins expressed by C. botulinum.
19. The method according to claim 1, wherein the purified botulinum
neurotoxin is 50-fold to 95-fold less toxic with respect to oral
ingestion by test animals than botulinum toxin complexed with
native accessory proteins expressed by C. botulinum.
20. The method according to claim 1, wherein the purified botulinum
neurotoxin is 30-fold less toxic with respect to oral ingestion by
test animals than botulinum toxin complexed with native accessory
proteins expressed by C. botulinum.
21. The method according to claims 18-20, wherein the test animals
are guinea pigs.
22. The method according to claims 18-20, wherein the test animals
are rats.
Description
FIELD OF THE INVENTION
[0001] This invention relates to compositions and methods for
treating rhinitis using topical botulinum toxin compositions. More
particularly, the invention relates to treating the symptoms of
rhinitis using intranasal administration of topical botulinum toxin
compositions that contain a purified botulinum neurotoxin.
BACKGROUND OF THE INVENTION
[0002] Rhinitis is a worldwide health problem associated with nasal
inflammation and characterized by symptoms of congestion,
rhinorrhea, sneezing and itching. Allergic rhinitis is the most
common form of rhinitis and affects up to 30% of adults and 40% of
children in the United States. Symptoms of both allergic as well as
non-allergic types of rhinitis can significantly impair patients'
quality of life. Moreover, allergic rhinitis often coexists with
other atopic conditions, such as asthma, sinusitis and sleep apnea.
Rhinitis is induced by overstimulation of parasympathetic
innervation of the nasal mucosal tissue via release of
acetylcholine and inflammatory mediators such as vasoactive
intestinal peptide (VIP). Pharmacologic therapy (e.g.,
anti-histamines, decongestants, corticosteroids, anti-cholinergics,
etc.) and in the case of allergic rhinitis, allergic immunotherapy,
require either frequent (one or more times per day) administration
often with side effects or a long-term process of desensitization
with limited effectiveness for many patients.
[0003] Botulinum toxin type A is obtained from serotype A of
Clostridium botulinum. Botulinum toxin type A acts to block the
release of acetycholine from the presynaptic nerve terminal with
consequent induction of muscular paralysis. Based on this
anti-cholinergic activity, botulinum toxin type A has been used
widely in the treatment of muscle spasticity disorders, and for the
cosmetic treatment of frown lines and wrinkles. In addition to
these applications in the neuromuscular system, botulinum toxin
type A blocks parasympathetic cholinergic transmission and has been
used for treatment of glandular hypersecretory disorders, such as
hyperhidrosis, Frey's syndrome, sialorrhea, epiphora, rhinorrhea,
and sialadenitis. It has also been shown that botulinum toxin type
A can be used to suppress electrically stimulated rhinorrhea in a
dog model, which is consistent with recent reports demonstrating
the effect of muscarinic cholinergic agents on canine nasal veins.
Subsequent studies in animal models and in human patients with
different types of rhinitis demonstrated that local application of
botulinum toxin type A effectively reduced rhinitis symptoms. In
humans, one botulinum toxin type A treatment injected directly into
the nasal turbinates significantly reduced rhinorrhoea for four
weeks (Kim K S, Kim S S, Yoon J H et al. The effect of botulinum
toxin type A injection for intrinsic rhinitis. J Laryngol Otol.
112:248-51, 1998) to eight weeks (Unal M, Sevim S, Do{hacek over
(g)}u O et al. Effect of botulinum toxin type A on nasal symptoms
in patients with allergic rhinitis: a double-blind,
placebo-controlled clinical trial. Acta Otolaryngol. 123:1060-3,
2003), and provided better symptom relief than corticosteroid
therapy for 20 weeks (Yang T Y, Jung Y G, Kim Y H et al. A
comparison of the effects of botulinum toxin A and steroid
injection on nasal allergy. Otolaryngol Head Neck Surg. 139:367-71,
2008). In addition, treatment of rhinitis using clostridial
neurotoxins has been reported in U.S. Pat. Nos. 5,766,605;
7,494,661; 7,879,340; 8,088,360; and 8,088,361, as well as in U.S.
Pre-Grant Publication Nos: 20120071395, 20110150975, 20110091505,
20110091504, 20110086072, 20110054442, 20060153876, 20060008462,
and 20040248188. Some of these references report delivering topical
liquid formulations containing commercially available botulinum
type A complexes by saturating a sponge or gauze packing with a
topical botulinum toxin formulation and inserting the sponge or
gauze packing into the nasal cavity. The practical difficulties
associated with this methodology raises certain procedural issues
as well as safety concerns. In addition to the treatment of
rhinitis, clostridial neurotoxins have been used in the treatment
of sinusitis (see, e.g. U.S. Pat. No. 8,092,781, WO2011/084507).
These and all other references cited herein are hereby incorporated
by reference in their entirety.
[0004] Despite this early evidence of efficacy, the use of
botulinum toxin type A for treating rhinitis has not been widely
accepted among clinicians and patients. One reason for this is the
oral toxicity of commercially available botulinum toxin
formulations. Botulinum toxin type A, in its native form, exists as
part of a protein complex that is produced by C. botulinum
bacteria. The native protein complex contains, in addition to the
botulinum toxin type A neurotoxin molecule, stabilizing
hemaglutinin and non-hemaglutinin proteins (sometimes referred to
as "accessory proteins") which are capable of protecting the
botulinum neurotoxin molecule from degradation in the harsh acidic
environment of the stomach. Currently available commercial
botulinum toxin type A formulations are made using native botulinum
toxin protein complexes. Thus, in the event of accidental oral
ingestion during the treatment of rhinitis, the accessory proteins
will enable the botulinum toxin to be passed through the stomach to
the small intestine, where it is absorbed into the bloodstream. If
this occurs, systemic poisoning, paralysis, and even death result.
Accordingly, treatment of rhinitis using the presently available
commercial botulinum toxin formulations in topical preparations has
been disfavored, even though botulinum toxin has been reported to
be able to bind to mucosal epithelial cells and become trancytosed
across mucosa. (See, e.g., U.S. Pat. No. 8,092,781).
[0005] While botulinum toxin has been reported to cross mucosa in
the respiratory and gastrointestinal tract, botulinum toxin does
not penetrate intact skin, owing to its size. See, e.g., S. Arnon
et al., "Botulinum Toxin as a Biological Weapon--Medical and Public
Health Management," JAMA, Vol. 285, No. 8, p. 1059 ff. Accordingly,
unlike delivering botulinum toxin to mucosa, one must take special
measures to either disrupt the skin (see, e.g., U.S. Pre-Grant
Publication No. 20070088248) or to enhance penetration of botulinum
toxin via use of a carrier (see, e.g., U.S. Pat. No. 7,807,780, and
U.S. Pre-Grant Publication Nos. 20050196414, 20070077259) when
administering botulinum toxin transdermally.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention relates to a method for
treating rhinitis. The method involves intranasally administering a
botulinum toxin composition to a patient in need of treatment for
rhinitis. In certain embodiments, the botulinum toxin composition
is a topical composition that includes (i) purified botulinum toxin
neurotoxin (ii) at least one carrier, and (iii) at least one
viscosity modifier. In such embodiments, the botulinum toxin is a
150 kDa botulinum neurotoxin type A which contains little or no
toxin accessory proteins or human/animal-derived components. Also,
the carrier comprises a positively charged backbone with positively
charged efficiency groups covalently attached thereto, and the
viscosity modifier is a preferably a poloxamer. Preferably, the
carrier and viscosity modifier help to retain the intranasal
composition at the site of administration and minimize the
possibility of drainage or leakage to other sites, e.g. down the
throat.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1.: Graph indicating performance severity assessment
score (PSA) of rats (mean.+-.SEM) on days 3, 5 and 7 after
treatment with control (filled bars) or reconstituted Formulation
22 (open bars).
[0008] FIG. 2. Effect of reconstituted Formulation 22 on
inflammatory pathology associated with allergic rhinitis in nasal
tissue. Histological staining of corresponding region of (a-c) left
turbinate and (d-f) lateral nasal wall from three animals, (a,d)
normal animal, (b, e) allergic control, and (c, f) allergic
reconstituted Formulation 22 treated, respectively. The thickness
of nasal mucosa was greater in (b, e) control animals than in (a,
d) normal and (c, f) reconstituted Formulation 22-treated allergic
animals. Remarkable mucosal edema, congestion and vascular
dilatation were noted in (b, e) control animals when compared with
(a, d) normal animals and (c, f) reconstituted Formulation
22-treated animals. (c, f) The reconstituted Formulation 22-treated
animals showed essentially normal nasal mucosal tissue with only
mild congestion (hematoxylin and eosin [H&E] stain; original
magnification, x20).
[0009] FIG. 3: Immunohistochemical localization of vasoactive
intestinal peptide in nasal turbinate. Serial sections from tissues
shown in FIG. 2, a-c, were prepared and stained for vasoactive
intestinal peptide (VIP) expression (indicated by dark staining)
Strong VIP expression was noted in (b) control animals when
compared with (a) normal animals. (c) After reconstituted
Formulation 22 treatment, VIP expression was down-regulated
essentially to normal levels (original magnification,
.times.20).
[0010] FIG. 4: Graph indicating effects of botulinum neurotoxin
type A (BoNTA) complex (900 U, ), reconstituted Formulation 27
(27,500 U, .tangle-solidup.) and saline control (.box-solid.) on
mean daily weight of guinea pigs. Animals received a single
intranasal dose of reconstituted Formulation 27, 27,500 U (OE);
BoNTA complex, 900 U (F); or saline control (f). Each symbol
represents the average of three animals with SD shown by error
bars. Asterisks denote values for reconstituted Formulation 27 that
were significantly reduced compared with same day values for
control (p<0.05).
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention is based, at least in part, on the
discovery that certain botulinum toxin compositions need not be
administered by injection for the treatment of rhinitis. The
botulinum toxin compositions according to the invention can be
administered by intranasally applying a topical botulinum toxin
formulation. It is believed that the present topical botulinum
toxin compositions offer at least two advantages over the
injectable botulinum toxin formulations in the prior art. First,
the present compositions utilize purified botulinum neurotoxin that
is substantially free of the accessory proteins, rather than
botulinum toxin complexes as used in conventional injectable
formulations. Without its native accessory proteins, botulinum
neurotoxin is susceptible to degradation in a patient's
gastrointestinal tract. Accordingly, the present topical botulinum
toxin compositions are far less likely to cause systemic poisoning
and death in the event that accidental oral ingestion occurs. In
addition, the at least one carrier and at least one viscosity
modifier help to prevent migration of the topical composition from
the site of administration, thereby minimizing the possibility of
migration and inadvertent oral ingestion. Without wishing to be
limited by theory, it is believed that the carrier contributes to
the localization of the botulinum toxin by facilitating transport
of the toxin across the mucosa to its site of action and minimizing
unwanted diffusion of the botulinum toxin away from the area in
need of treatment. Moreover, it is believed that the viscosity
modifier contributes to creating a formulation that essentially
stays in the location where it is applied. Accordingly, the topical
botulinum toxin compositions of the present invention provide an
improved safety profile over the previous injectable botulinum
toxin compositions.
[0012] The invention is suitable for the treatment of all forms of
rhinitis, non-limiting examples of which include infectious
rhinitis, vasomotor rhinitis, allergic rhinitis, rhinitis
medicamentosa, atrophic rhinitis, rhinitis sicca, and polypous
rhinitis. The compositions and methods disclosed herein can be used
to treat rhinitis or characteristic symptoms of the same, e.g.
nasal inflammation, nasal congestion, rhinorrhea, sneezing and/or
itching. Thus, for example, the invention may be used to treat
rhinitis-like symptoms, such as those that occur following certain
ear-nose-throat (ENT) procedures, a non-limiting example of which
is rhinoplasty. When the invention is being used to treat rhinitis,
the rhinitis can be allergic rhinitis or non-allergic rhinitis,
where the latter type of rhinitis can be either inflammatory or
non-inflammatory rhinitis (e.g. vasomotor rhinitis). It is also
contemplated that multiple forms of rhinitis can be treated
simultaneously with the compositions and methods of the present
invention. For example, the compositions and methods of the present
invention can be used to treat a patient suffering from both
allergic rhinitis and vasomotor rhinitis, a condition that is
commonly referred to as "mixed rhinitis." The methods can also be
used to treat rhinitis when it occurs in patients who have a
condition in combination with other atopic conditions, such as
asthma, sinusitis and sleep apnea.
[0013] Generally, the topical botulinum toxin compositions
contemplated by the invention can be administered using an
applicator that is inserted into a patients' nostril. Optionally,
the topical administration may be achieved using visual guidance
with the assistance of a nasal speculum and/or headlamp as
necessary. The topical composition is applied with the applicator
to inner surfaces of the nose, non-limiting examples of which
include the surfaces of the inferior turbinate, middle turbinate
and the superior turbinate. The topical botulinum toxin
compositions may be administered via one nostril or via both
nostrils, if deemed necessary. Application of the topical
composition may be achieved, for example, by using a custom
applicator, such as the one described in U.S. Pre-Grant Publication
No. 2011010621 to Ruegg et al., which is hereby incorporated by
reference in its entirety. Alternatively, the topical composition
may be applied first to an implement, such as a swab, which is then
used to spread the topical composition over the area in need of
treatment.
[0014] After the topical botulinum toxin compositions of the
invention are administered, they optionally may be allowed to
remain in place for a certain dwell period in order to increase the
amount of botulinum neurotoxin that is delivered to the nasal
tissues in need of treatment. As will be appreciated by those of
skill in the art, the specific dwell time that is selected will
depend on factors such as the severity of the rhinitis, the desired
amount of botulinum toxin to be delivered, the concentration of the
botulinum neurotoxin in the topical botulinum toxin composition,
the concentration of the carrier in the topical botulinum toxin
composition, and the viscosity of the topical botulinum toxin
composition. Optionally, the dwell times may range from 5 seconds
to 60 minutes, 30 seconds to 45 minutes, 1 minute to 30 minutes, 5
minutes to 20 minutes or 10 minutes to 15 minutes. Optionally, the
dwell time may be 10, 20, 30, 40 or 50 minutes. After the chosen
dwell time has elapsed, the excess topical botulinum toxin
composition on the inner surfaces of the nose, to the extent that
any such excess exists, may be removed by any suitable means,
non-limiting examples of which including swabbing the inner
surfaces of the nose or flushing the nostrils with a liquid, such
as saline.
Purified Botulinum Toxin
[0015] The term "purified botulinum neurotoxin," as used herein,
refers to any of the known types of purified botulinum neurotoxin,
whether produced by the bacterium or by recombinant techniques, as
well as any such types that may be subsequently discovered,
including engineered variants or fusion proteins. For example, the
purified botulinum neurotoxin may be a compound that has toxin
activity but contains one or more chemical or functional
alterations on any part or on any chain relative to naturally
occurring or recombinant native neurotoxins. The purified botulinum
neurotoxin may be a modified neurotoxin that has at least one of
its amino acids deleted, modified or replaced, as compared to a
native neurotoxin molecule while maintaining significant neurotoxin
activity. In this regard, modified botulinum neurotoxins obtained
from a native botulinum neurotoxin with one or more of its amino
acids replaced by conservative substitutions are expressly
contemplated by the invention. Alternatively, the purified
botulinum neurotoxin 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. The purified botulinum neurotoxin may be
prepared using recombinant or synthetic chemical techniques (e.g. a
recombinant peptide, a fusion protein, or a hybrid neurotoxin, as
prepared from subunits or domains of different botulinum toxin
serotypes (see, e.g., U.S. Pat. No. 6,444,209, the contents of
which are incorporated by reference in their entirety). The
purified botulinum neurotoxin may also be a portion of the overall
molecule that has been shown to possess the necessary botulinum
toxin neurotoxin activity, and in such case may be used per se or
as part of a combination or conjugate molecule, for instance a
fusion protein. Additionally, the purified botulinum neurotoxin may
be in the form of a purified botulinum neurotoxin precursor, which
may itself be non-toxic, for instance a nontoxic zinc protease that
becomes toxic on proteolytic cleavage.
[0016] Optionally, the purified botulinum neurotoxin is obtained by
isolating it from C. botulinum bacteria. In this regard, the
invention expressly contemplates the use of purified botulinum
neurotoxins selected from the group of clostridial neurotoxins
consisting of serotypes A, B, C1, D, E, F, G and combinations
thereof. In the compositions of the present invention, the purified
botulinum neurotoxin is present as an isolated botulinum neurotoxin
(e.g., purified botulinum toxin type A protein) that is stabilized
by exogenous stabilizers. Optionally, the compositions are
substantially free of the accessory proteins normally found in
native botulinum toxin complexes. Preferably, the botulinum
neurotoxin is sufficiently free of accessory proteins that the
neurotoxin is susceptible to degradation in the gastrointestinal
tract if ingested. For example, botulinum neurotoxin may be
substantially free of accessory proteins if at least 70%, 80%, 85%,
90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the accessory
proteins found in the native botulinum toxin complexes have been
removed, with each of the ranges from these specifically enumerated
lower limits to 100% being a distinct embodiment expressly
contemplated by the invention. Optionally, the purified botulinum
neurotoxin is entirely free of the neurotoxin-associated proteins
characteristic of the 900 kDa botulinum toxin complex. Stabilized
botulinum neurotoxin formulations containing such exogenous
stabilizers have been reported, for example, in U.S. Pre-Grant
Publication 20100330123 entitled "Albumin Free Botulinum Toxin
Formulations," which is hereby incorporated by reference in its
entirety.
[0017] The purified botulinum neurotoxins contemplated by the
invention are less toxic with respect to oral ingestion than native
botulinum toxin, owing to the removal of some or all of the
accessory proteins normally accompanying the native botulinum
neurotoxin. The invention contemplates removing a sufficient amount
of the native accessory proteins to obtain a purified botulinum
neurotoxin that is 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,
60-fold, 70-fold, 80-fold or 90-fold less toxic with respect to
oral ingestion than native botulinum toxin. For example, the
purified botulinum neurotoxin may be 10-fold to 90-fold less toxic,
20-fold to 80-fold less toxic, 30-fold to 70-fold less toxic, or
40-fold to 60-fold less toxic with respect to oral ingestion.
Optionally, the purified botulinum toxin may be 50-fold to 95-fold
less toxic, 60-fold to 90-fold less toxic, or 70-fold to 85-fold
less toxic with respect to oral ingestion than the native botulinum
toxin. The relative toxicities of botulinum neurotoxin-containing
formulations may be determined using test animal studies, as
described herein. Removal of the accessory proteins may be
accomplished using any methods known in the art, such as treating
the native botulinum toxin complexes with red blood cells at a pH
of 7.3, or by using separation methods such as chromatography (See,
e.g., U.S. Pre-Grant Publication No. 20110092682, the contents of
which are incorporated by reference in their entirety).
[0018] The purified botulinum neurotoxin is present in the topical
botulinum toxin compositions of the invention in an effective
amount to treat rhinitis. In this context, the term "effective
amount" refers to an amount of purified botulinum neurotoxin that
is sufficient to ameliorate one or more symptoms of rhinitis
safely. For example, when the purified botulinum neurotoxin is
serotype A, the amount of purified botulinum neurotoxin may range
from 250 U/mL to 50,000 U/mL, 500 U/mL to 25,000 U/mL, 2,500 U/mL
to 12,500 U/mL, or 5,000 U/mL to 10,000 U/mL. In general, effective
amounts for the purified botulinum neurotoxins disclosed herein may
range from 50 U/mL to 450,000 U/mL, preferably from 5,000
U/mL-400,000 U/mL, more preferably from 10,000 U/mL-150,000 U/mL,
and even more preferably from 20,000 U/mL to 125,000 U/mL.
Optionally, the effective amount of purified botulinum toxin may
range from 20,000 U/mL to 40,000 U/mL, 45,000 U/mL to 95,000 U/mL,
or 100,000 U/mL to 200,000 U/mL.
[0019] The invention also contemplates topical botulinum toxin
compositions that contain purified botulinum neurotoxin present in
the composition a concentration that ranges from about 5 ng/mL to
about 50 ng/mL, such as, for example about 5 ng/mL, about 10 ng/mL,
about 15 ng/mL, 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35
ng/mL, about 40 ng/mL, about 45 ng/mL or about 50 ng/mL. Of course,
the specific amount of purified botulinum neurotoxin in a given
topical formulation will depend on the chosen carrier and viscosity
modifier, and can be readily determined by a person of skill in the
art. Optionally, the concentration and volume of the topical
botulinum toxin composition are selected so as to provide a dose of
botulinum toxin in the range of 1 ng to 50 ng, or 5 ng to 40 ng, or
10 ng to 30 ng, or 15 ng to 25 ng. For example, if desired, the
dose of botulinum toxin can be in a range from 1 ng to 10 ng, 2 ng
to 8 ng, or 3 ng to 6 ng. The dose of botulinum toxin optionally
may be in the range of 0.5 ng to 5 ng, 1 ng to 5 ng, or 2 ng to 4
ng. The dose of botulinum toxin in certain preferred embodiments is
1, 2, 3, 4, or 5 ng. Optionally, the volume of the applied topical
botulinum toxin composition is in the range of 0.02-0.5 mL, 0.05 to
0.4 m, 0.07 to 0.3 mL or 0.09 to 0.2 mL, and the concentration of
the topical botulinum toxin is selected to provide a dose of 1 to
10 ng, or 1 to 5 ng.
Carriers
[0020] In addition to purified botulinum neurotoxin, the topical
botulinum toxin compositions of the invention preferably contains a
carrier to promote the transport of the purified botulinum
neurotoxin into the nasal tissues in need of treatment. In its most
general implementation, the invention contemplates the use of
carriers that promote the transport of botulinum toxin from applied
topical compositions. For example, the carrier may be a liquid
chemical carrier, such as dimethyl sulfoxide (DMSO). The carrier
may also be in the form of a nanoemulsion as described in
WO2008/045107. In other embodiments, the carrier may comprise
sphingosine and/or cerebroside as described in U.S. Pre-Grant
Publication No. 20060182766. The carrier may also comprise a
sialoprotein, such as those described, for example in U.S.
Pre-Grant Publication No. 20070116724. It is to be understood that
the foregoing references, like all references cited herein, are
incorporated by reference in their entirety.
[0021] Optionally, the topical compositions according to the
invention comprise a carrier that includes a positively charged
carrier molecule with positively charged efficiency groups attached
thereto, as is disclosed, for example, in U.S. Pat. No. 8,398,997,
which is hereby incorporated by reference in its entirety. By
"positively charged," it is meant that the carrier molecule has a
positive charge under at least some solution-phase conditions, more
preferably under at least some physiologically compatible
conditions. The term "positively charged," as used herein, embraces
functionalities that are charged under all pH conditions, for
instance, a quaternary amine, as well as functionalities which can
acquire positive charge under certain solution-phase conditions,
such as pH changes, in the case of primary amines. More preferably,
"positively charged" 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.
Without wishing to be limited by theory, it is believed that the
positively charged carriers of the invention help to
electrostatically anchor the botulinum toxin to the treated mucosa,
thereby reducing unwanted diffusion, reducing unwanted drainage of
the topical formulation into the throat or airway, and increasing
efficacy.
[0022] In certain embodiments, the positively charged carrier
molecule comprises a "positively charged backbone," which is
typically a linear 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. Preferably, the positively charged backbone itself
will not have a defined enzymatic or therapeutic biologic activity.
The linear backbone is preferably a 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. Generally, the
positively charged backbone will have a molecular weight that
ranges from 100-2,500,000, more preferably, 200-2,000,000, even
more preferably, 300-500,000, 400-100,000, 500-50,000, 600-20,000
or 700-8,000. Optionally, 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 500 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.
Optionally, the nonpeptidyl polymer may have a molecular weight in
the range from about 500 to about 5000, about 1000 to about 4000,
about 1500 to about 3500, or about 2000 to about 3000. If desired,
the backbone may have attached thereto 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. In all aspects of the present invention, the association
between the carrier and the chemodenervating agent is by
non-covalent interaction, non-limiting examples of which include
ionic interactions, hydrogen bonding, van der Waals forces, or
combinations thereof.
[0023] Optionally, the positively charged backbone is a polypeptide
having multiple positively charged sidechain groups (e.g., lysine,
arginine, ornithine, homoarginine, and the like). For instance, the
polypeptide may a molecular weight of from about 500 to about
1,500,000, about 10,000 to about 1,500,000, more preferably from
about 25,000 to about 1,200,000, most preferably from about 100,000
to about 1,000,000. If desired, the polypeptide may have a
molecular weight in the range from about 500 to about 5,000, about
1,000 to about 4,000, about 1,500 to about 3,500, or about 2,000 to
about 3,000. 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. Alternatively, the backbone can 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.
[0024] 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.
[0025] In each of the backbones provided above, sidechain groups
can be appended that carry a positively charged group, preferably
at physiologic pH. 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.
[0026] Optionally, the positively charged backbone is a polypeptide
having efficiency groups. As used herein, an efficiency group 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 efficiency groups include
-(gly).sub.n1-(arg).sub.n2, HIV-TAT or fragments thereof, or the
protein transduction domain 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 a preferred embodiment n1 is 3 and n2 is 7. Still
further preferred are those embodiments in which the efficiency
group has the formula (gly).sub.p-RGRDDRRQRRR-(gly).sub.q,
(gly).sub.p-YGRKKRRQRRR-(gly).sub.q or
(gly).sub.p-RKKRRQRRR-(gly).sub.q wherein the subscripts p and q
are each independently an integer of from 0 to 20 and the fragment
is attached to the backbone via either the C-terminus or the
N-terminus of the fragment. Preferred efficiency groups are those
in which the subscripts p and q are each independently integers of
from 0 to 8, more preferably 2 to 5. In some embodiments, the
carrier has the amino acid sequence selected from the group
consisting of RKKRRQRRR-G-(K).sub.15-G-RKKRRQRRR,
RKKRRQRRR-G-(K).sub.20-G-RKKRRQRRR,
RKKRRQRRR-G-(K).sub.25-G-RKKRRQRRR,
RKKRRQRRR-G-(K).sub.30-G-RKKRRQRRR,
RGRDDRRQRRR-G-(K).sub.15-G-RGRDDRRQRRR,
RGRDDRRQRRR-G-(K).sub.20-G-RGRDDRRQRRR,
RGRDDRRQRRR-G-(K).sub.25-G-RGRDDRRQRRR,
RGRDDRRQRRR-G-(K).sub.30-G-RGRDDRRQRRR,
YGRKKRRQRRR-G-(K).sub.15-G-YGRKKRRQRRR,
YGRKKRRQRRR-G-(K).sub.20-G-YGRKKRRQRRR,
YGRKKRRQRRR-G-(K).sub.25-G-YGRKKRRQRRR, and
YGRKKRRQRRR-G-(K).sub.30-G-YGRKKRRQRRR. See, e.g., U.S. Pat. No.
8,404,249, and U.S. Pre-Grant Publication No. 20100215591, both of
which are incorporated by reference in their entirety.
[0027] In another preferred embodiment the positively charged
efficiency group is the Antennapedia (Antp) protein transduction
domain (PTD), or a fragment thereof that retains activity. (See,
e.g., Console et al., J. Biol. Chem. 278:35109 (2003), the contents
of which are incorporated by reference in their entirety.)
Preferably the positively charged carrier includes side-chain
positively charged efficiency groups in an amount of at least about
0.05%, as a percentage of the total carrier weight, preferably from
about 0.05 to about 45 weight %, and most preferably from about 0.1
to about 30 weight %. For positively charged efficiency groups
having the formula -(gly).sub.n1-(arg).sub.n2, the most preferred
amount is from about 0.1 to about 25%.
[0028] In certain embodiments, the backbone portion is a polylysine
and positively charged efficiency groups are attached to the lysine
sidechain amino groups. In some embodiments, the polylysine may
have a molecular weight that ranges from about 10,000 to about
1,500,000, preferably from about 25,000 to about 1,200,000, and
most preferably from about 100,000 to about 1,000,000. In certain
embodiments, the polylysine may have a molecular weight that ranges
from about 500 to about 5000, about 1000 to about 4000, about 1500
to about 3500, or about 2000 to about 3000. The polylysine may 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, in some embodiments, provide a length that is
preferably from one to four times the combined length of the
negatively charged components. Preferred positively charged
efficiency groups or efficiency groups include, for example,
-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly.sub.3Arg.sub.7) or
HIV-TAT fragments, as disclosed herein. In another preferred
embodiment the positively charged backbone is a long chain
polyalkyleneimine such as a polyethyleneimine or
polypropyleneimine. Such polyalkyleneimines, for example, may have
a molecular weight of about 1,000,000.
[0029] In certain 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. 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, more preferably less than
30,000, and most preferably, less than 25,000. When the carrier is
a relatively short branched polylysine or PEI backbone, however,
the backbone will have a molecular weight less than 60,000, more
preferably less than 55,000, and most preferably less than 50,000.
For example, if desired, the backbone may have a molecular weight
that ranges from about 500 to about 5000, about 1000 to about 4000,
about 1500 to about 3500, or about 2000 to about 3000.
Viscosity Modifiers
[0030] The topical botulinum toxin compositions for treating
rhinitis according to the methods described herein typically also
include a viscosity modifier to maintain the location of the
applied compositions at the treatment area, to restrict the
movement of the compositions from the intended treatment area after
application, and/or to minimize or prevent diffusion and
inadvertent oral and/or systemic exposure. Generally, the chemical
identity of the viscosity modifier is not particularly limited, and
may be any pharmaceutically acceptable composition with the
appropriate viscosity at the body surface temperature at the area
of application, so long as the viscosity modifier is compatible
with other components of the topical botulinum toxin compositions,
as described herein.
[0031] The viscosity modifier according to the invention may be
chosen such that the viscosity of the topical botulinum toxin
composition falls within a viscosity range of 10,000-500,000 cps,
more preferably, 15,000-250,000 cps, even more preferably 20,000 to
200,000 cps and most preferably 25,000 to 100,000 cps at 25.degree.
C. Optionally, the viscosity modifier may be chosen such that the
viscosity of the topical botulinum toxin composition is
.gtoreq.1000 cps at 26.degree. C., such as, for example, from
1,000-10,000 cps, 1,500-8,000 cps, 2,000-6,000 cps, or 2,500-5,000
cps. The required amount of viscosity modifying agent can be
determined readily by a person of skill in the art, based on the
disclosures set forth herein.
[0032] The viscosity modifier optionally may be a surfactant. The
surfactant may be selected from anionic surfactants, cationic
surfactants, zwitterionic surfactants or non-ionic surfactants. In
certain embodiments, one or more non-ionic surfactants serve as the
viscosity modifier. The non-ionic surfactant can be any
commercially available non-ionic surfactant, such as, for example,
polyoxyethylene glycol alkyl ethers, polyoxypropylene glycol alkyl
ethers, glucoside alkyl ethers, polyoxyethylene glycol octylphenol
ethers, polyoxyethylene glycol alkylphenol ethers, glycerol alkyl
esters, polyoxyethylene glycol sorbitan alkyl esters, sorbitan
alkyl esters, dodecyldimethylamine oxide, block copolymers of
polyethylene glycol and polypropylene glycol (polyoxamers) and
combinations thereof.
[0033] In certain embodiments, the non-ionic surfactant is a
polysorbate, non-limiting examples of which include 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 20, Span 60, Span 65,
and Span 80. The invention also contemplates using Triton X-100,
trileucine, or NP-40 as the non-ionic surfactants. In addition, the
combinations of different non-ionic surfactants are contemplated.
In certain preferred embodiments, the non-ionic surfactant is
selected from the group consisting of polysorbates, poloxamers, and
sorbitans, with polysorbates and sorbitans being particularly
preferred.
[0034] In a particular embodiment, the viscosity modifier can be a
poloxamer. Such poloxamers may be linear or branched, and include
tri-blocks or tetra-blocks copolymers. They include poloxamines
such as Tetronic and Pluronic. The poloxamer may be chosen from
poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122,
poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182,
poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188,
poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231,
poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238,
poloxamer 282, poloxamer 284, poloxamer 288, poloxamer 331,
poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338,
poloxamer 401, poloxamer 402, poloxamer 403, poloxamer 407, and
combinations thereof. In certain preferred embodiments, the
poloxamer that is chosen has a tendency to form a gel with
increasing temperature. Non-limiting examples of such poloxamers
include poloxamer 188 and poloxamer 407.
[0035] As the skilled artisan will appreciate, the amount of
viscosity modifying agent that is present in the topical botulinum
toxin compositions of the invention will depend on the identity of
the viscosity modifying agent, as well as the desired viscosity of
the topical botulinum toxin composition. With this in mind,
suitable concentrations for viscosity modifying agent in the
topical botulinum toxin compositions of the invention may range
from about 5% and about 70% (wt/wt), such as, for example, between
about 5% and about 60%, between about 10% and about 50%, between
about 15% and about 40%. Optionally, the viscosity modifier is
present in the compositions in concentration between about 15% and
about 20%, such as, for example, about 16%, about 17%, about 18%,
about 19% or about 20%. Optionally, the viscosity modifying agent
is present in a concentration of 15.0%, 15.5%, 16.0%, 16.5%, 17.0%,
17.5%, 18.0%, 18.5%, 19.0%, 19.5%, or 20% and is selected from the
group consisting of poloxamer 212, poloxamer 215, poloxamer 217,
poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237,
poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288,
poloxamer 331, poloxamer 333, poloxamer 334, poloxamer 335,
poloxamer 338, poloxamer 401, poloxamer 402, poloxamer 403,
poloxamer 407, and combinations thereof. For example, in certain
preferred embodiments, when the poloxamer is poloxamer 407, the
amount of poloxamer present in the formulations of the invention
may range from 15-25%, 15.5-24.5%, 16-23%, 16.5-22.5%, 17-22%
17.5%-21.5%, or 18%-21%. Optionally, the amount of poloxamer 407
may be 15.5%, 16.0%, 16.5%, 17.0%, 17.5%, 18.0%, 18.5%, 19.0%,
19.5%, 20%, 20.5%, 21% 21.5% or 22%. Alternatively, the chosen
poloxamer may be poloxamer 188, which may be present in the
formulations of the invention at a concentration range of 15.5%,
16.0%, 16.5%, 17.0%, or 17.5%. This invention also specifically
contemplates adding more than one type of poloxamer to modify the
viscosity of the formulation. For instance, if desired, both
poloxamer 188 and poloxamer 407 may be added to the formulations of
the invention to modify the viscosity.
[0036] In addition, the topical formulations according to the
present invention may also comprise stabilizing agents. For
example, such stabilizing agents may include non-reducing sugars,
non-limiting examples of which include trehalose and sucrose. In
preferred embodiments, the non-reducing sugar has a glass
transition temperature above 55.degree. C., 57.degree. C., or
60.degree. C. Without wishing to be bound by theory, it is believed
that such glass transition temperatures are sufficiently high to
suppress undesirable molecular motions that cause the botulinum
toxin to denature. In certain particularly preferred embodiments,
the non-reducing sugar is a disaccharide, non-limiting examples of
which include trehalose and sucrose. In other embodiments, the
non-reducing sugar is a trisaccharide, a non-limiting example of
which is raffinose. Generally, the concentration of the
non-reducing sugar in the botulinum toxin formulations 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 is 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% (w/v). When the
non-reducing sugar is trehalose, preferably the hydrated form of
trehalose (i.e., trehalose-dihydrate) is used to prepare the
formulation, although use of the anhydrous form of trehalose is
contemplated as well. Since the formulations of the present
invention are typically lyophilized, an agent may be added to
increase the mechanical strength of amorphous glass solid cake that
is formed when the formulation is lyophilized. A non-limiting
example of such an agent includes boric acid.
[0037] Optionally, the topical botulinum toxin formulations of the
invention comprise a buffer. Generally, any physiologically
compatible buffer capable of maintaining the pH in the range of 4.5
to 6.5, more preferably in the range of 5 to 6, and most preferably
about 5.5, is suitable for the botulinum toxin formulations of the
invention. Non-limiting examples of such buffers include those
involving 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 invention also contemplates botulinum toxin formulations
comprising a mixture of buffer salts, non-limiting examples of
which include citrate/acetate, citrate/histidine, citrate/tartrate,
maleate/histidine, or succinate/histidine. In certain preferred
embodiments, the buffer is phosphate buffer.
[0038] The topical botulinum formulations may also comprise an
anti-oxidant that acts as a preservative. Non-limiting examples of
such preservatives include butylhydroxytoluene (BHT), methionine,
and propyl gallate. One or more of such preservatives may be
present in the topical botulinum toxin formulations of the
invention in a concentration that ranges from 0.3-2.0%, from
0.5%-1.5%, or from 0.75%-1.25%.
Treatment Regimens
[0039] The methods described herein can be incorporated into
various treatment regimens. For example, the methods for treating
rhinitis described herein may be performed on a given patient every
one week, two weeks, three weeks, month, two months, three months,
four months, five months, six months, seven months, eight months,
nine months, ten months, eleven months or twelve months. The length
of time between applications will vary depending on many factors,
including the specific purified botulinum toxin chosen, the dosage
(which depends on the concentration of topical composition and the
dwell time) and the severity of the rhinitis. As a non-limiting
example, purified botulinum toxin type A may be administered every
three or four months for treating rhinitis, as needed.
[0040] Optionally, the topical compositions having a botulinum
toxin concentration of 1,000-50,000 U/mL may be administered. For
example, compositions having a concentration of 2,000 U/mL, 3000,
U/mL, 4,000 U/mL, 5,000 U/mL, 6,000 U/mL, 7,000 U/mL, 8,000 U/mL or
9,000 U/mL may be topically applied to the nasal cavity as
described herein for a dwell time of 10, 20, 30, 40, or 50 minutes.
If desired, topical compositions having a botulinum toxin
concentration of 5,500 U/mL, 5,750 U/mL, 6,000 U/mL, 6,250 U/mL,
6,500 U/mL, 6,750 U/mL or 7,000 U/mL may be topically applied to
the nasal cavity as described herein for a dwell time of 10, 20,
30, 40, or 50 minutes. Alternatively, topical compositions having a
botulinum toxin concentration of 15,500 U/mL, 15,750 U/mL, 16,000
U/mL, 16,250 U/mL, 16,500 U/mL, 16,750 U/mL or 17,000 U/mL may be
topically applied to the nasal cavity as described herein for a
dwell time of 10, 20, 30, 40, or 50 minutes. One particularly
suitable treatment involves applying 0.2 mL of a topical botulinum
toxin type A composition containing 6,250 U/mL for a dwell time of
30 minutes. After the dwell time has elapsed, the topical botulinum
toxin may be flushed out of the patient's nasal cavity by
irrigating the nasal cavity with saline solution. As the skilled
artisan will appreciate, the resulting effluent contains botulinum
toxin and is highly toxic. Preferably, the effluent is collected
(e.g., absorbed by an absorbent article) and the botulinum toxin
therein is reacted with an agent to render it no longer toxic.
Suitable methods and agents for inactivating botulinum toxin
include those found, for example, in U.S. patent application Ser.
No. 13/334,283, which is hereby incorporated by reference in its
entirety.
Formulations
[0041] The topical formulations of the invention optionally may be
manufactured in a lyophilized form and then reconstituted with a
diluent before administration. For example, the lyophilized form
may be produced by lyophilizing a liquid composition comprising a
botulinum toxin, a non-reducing disaccharide or a non-reducing
trisaccharide, a non-ionic surfactant, and a physiologically
compatible buffer. Typically, in such formulations, the
concentration of the non-reducing disaccharide or non-reducing
trisaccharide is in the range of 10% to 40% (w/v), the
concentration of the non-ionic surfactant is in the range of 0.005%
to 0.5% (w/v), and the pH of the liquid composition is in the range
of 4.5 to 6.5. See, e.g., U.S. Pre-Grant Publication No.
20100330123, which is hereby incorporated by reference in its
entirety. Generally, any pharmaceutically acceptable diluent that
does not undergo undesirable reactions with the components of the
formulation in question may be used. For example, the formulations
may be reconstituted using water, saline, or phosphate buffered
saline. Optionally, one or more additives may be included in the
diluent to control or improve certain properties of the diluent,
non-limiting examples of which include viscosity enhancers (e.g., a
poloxamer, such as poloxamer 188 or 407), anti-oxidants (e.g., BHT
or methionine), co-solvents (e.g., an alcohol, such as ethanol),
and/or tonicity adjusters (e.g., a salt, such as sodium chloride).
Exemplary diluents that are suitable for use with the invention
include those listed in Tables 3 and 3-1, as set forth herein. For
lyophilized formulations that are stored in 2 ml vials, it is often
convenient to reconstitute the formulations using 1 ml of diluent,
non-limiting examples of which include those shown in Tables 3 and
3-1.
EXAMPLES
Example 1
Topical Formulations
[0042] Table 1 shows 35 exemplary lyophilized topical botulinum
toxin formulations that were prepared in accordance with the
present invention. Each formulation was prepared by adding the
respective components in the indicated amounts to a standard 2 ml
lyophilization vial. The column heading "Toxin (ng/vial)" refers to
the amount of botulinum toxin type A neurotoxin present (in
nanograms per vial), while the column heading "peptide (mg/ml)"
refers to the amount of the peptide
RKKRRQRRR-Q-(K).sub.15-Q-RKKRRQRRR present (in milligrams per
vial). Table 1-1 shows the same 35 exemplary lyophilized topical
botulinum toxin formulations as in Table 1, except that the
numerical entries in Table 1-1 refer to the respective weight
percent of the components relative to the total weight of the
lyophilized formulation.
TABLE-US-00001 TABLE 1 Exemplary topical botulinum toxin
formulations Histidine Toxin Peptide Sodium Citrate Histidine
Hydrochloride Sucrose Trehalose Formulation (ng/vial) (mg/vial)
dehydrate (mg/vial) (mg/vial) (mg/vial) (mg/vial) (mg/vial) 1 11.0
0.45 0.10 20.0 2 11.0 0.030 0.45 0.10 20.0 3 11.0 0.030 0.45 0.10 4
11.0 0.030 0.45 0.10 36.0 5 11.0 0.030 0.14 0.65 36.0 6 11.0 0.010
0.14 0.65 36.0 7 11.0 0.003 0.14 0.65 36.0 8 11.0 0.030 0.14 0.65
8.0 9 11.0 0.030 0.45 0.10 36.0 10 11.0 0.030 0.45 0.10 11 11.0
0.030 0.45 0.10 8.0 12 11.0 0.030 0.45 0.10 34.0 13 11.0 0.030 0.45
0.10 36.0 14 11.0 0.030 0.45 0.10 36.0 15 1.1 0.030 0.14 0.65 36.0
16 1.1 0.030 0.45 0.10 36.0 17 1.1 0.030 0.14 0.65 8.0 18 1.1 0.030
0.14 0.65 4.0 19 1.5 0.009 0.45 0.10 36.0 20 3 0.009 0.45 0.10 36.0
21 6 0.009 0.45 0.10 36.0 22 10 0.009 0.45 0.10 36.0 23 12 0.009
0.45 0.10 36.0 24 25 0.009 0.45 0.10 36.0 25 36 0.009 0.45 0.10
36.0 26 50 0.009 0.45 0.10 36.0 27 5.5 .times. 10.sup.3 0.009 0.45
0.10 36.0 28 1.1 .times. 10.sup.4 0.009 0.45 0.10 36.0 29 2.2
.times. 10.sup.4 0.009 0.45 0.10 36.0 30 1.03 .times. 10.sup.4
0.009 0.45 0.10 36.0 31 2.06 .times. 10.sup.4 0.009 0.45 0.10 36.0
32 4.13 .times. 10.sup.4 0.009 0.45 0.10 36.0 33 5.87 .times.
10.sup.4 0.009 0.45 0.10 36.0 34 8.25 .times. 10.sup.4 0.009 0.45
0.10 36.0 35 1.76 .times. 10.sup.5 0.009 0.45 0.10 36.0 Methionine
BHT Poloxamer Poloxamer Trileucine Polysobate Formulation (mg/vial)
(mg/vial) 188 (mg/vial) 407 (mg/vial) (mg/vial) 20 (mg/vial) 1 10.0
0.2 2 10.0 0.2 3 36.0 0.10 4 0.10 5 6 7 8 28.0 2.0 9 10 36.0 11
28.0 12 13 0.2 14 0.20 15 16 17 28.0 18 14.0 19 0.10 20 0.10 21
0.10 22 0.10 23 0.10 24 0.10 25 0.10 26 0.10 27 0.10 28 0.10 29
0.10 30 0.10 31 0.10 32 0.10 33 0.10 34 0.10 35 0.10
TABLE-US-00002 TABLE 1-1 Exemplary topical botulinum toxin
formulations (amounts expressed in terms of weight percent relative
to total weight of the lyophilized formulation) Sodium His-
Histidinie Formu- Toxin Citrate ti- Hydrochlo- Treha- Methi-
Poloxamer Poloxamer Polysor- lation (.times.10.sup.-5) Peptide
dehydrate dine ride Sucrose lose onine BHT 188 407 Trileucine bate
20 1 3.6 1.5 0.3 65.0 32.5 0.7 0.0 2 3.6 0.1 1.5 0.3 65.0 32.5 0.6
0.0 3 4.1 0.1 1.7 0.4 97.5 0.4 0.0 0.0 4 3.0 0.1 1.2 0.3 98.1 0.3
0.0 0.0 5 3.0 0.1 0.4 1.8 97.5 0.0 0.0 0.0 6 3.0 0.1 0.4 1.8 97.5
0.0 0.0 0.0 7 3.0 0.1 0.4 1.8 97.5 0.0 0.0 0.0 8 2.8 0.1 0.4 1.7
20.6 71.9 5.1 0.0 0.0 0.0 9 3.0 0.1 1.2 0.3 98.1 0.0 0.0 0.0 10 4.1
0.1 1.7 0.4 0.0 97.5 0.0 0.0 0.0 11 3.0 0.1 1.2 0.3 21.8 76.3 0.0
0.0 0.0 12 3.2 0.1 1.3 0.3 98.0 13 3.0 0.1 1.2 0.3 97.6 0.5 14 3.0
0.1 1.2 0.3 97.9 0.5 15 3.0 0.1 0.4 1.8 97.5 16 3.0 0.1 1.2 0.3
98.1 17 4.8 0.1 0.6 2.8 34.9 61.1 18 2.2 60.7 1.3 1.3 8.1 28.3 19
0.41 2.5 .times. 10.sup.-5 1.2 0.3 98.2 0.3 20 0.82 2.5 .times.
10.sup.-5 1.2 0.3 98.2 0.3 21 1.6 2.5 .times. 10.sup.-5 1.2 0.3
98.2 0.3 22 2.7 2.5 .times. 10.sup.-5 1.2 0.3 98.2 0.3 23 3.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 24 6.8 2.5 .times. 10.sup.-5 1.2
0.3 98.2 0.3 25 9.8 2.5 .times. 10.sup.-5 1.2 0.3 98.2 0.3 26 14
2.5 .times. 10.sup.-5 1.2 0.3 98.2 0.3 27 1.5 .times. 10.sup.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 28 3.0 .times. 10.sup.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 29 6.0 .times. 10.sup.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 30 2.8 .times. 10.sup.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 31 5.6 .times. 10.sup.3 2.5
.times. 10.sup.-5 1.2 0.3 98.2 0.3 32 1.1 .times. 10.sup.4 2.5
.times. 10.sup.-5 1.2 0.3 98.1 0.3 33 1.6 .times. 10.sup.4 2.5
.times. 10.sup.-5 1.2 0.3 98.1 0.3 34 2.2 .times. 10.sup.4 2.5
.times. 10.sup.-5 1.2 0.3 98.0 0.3 35 2.2 .times. 10.sup.4 2.5
.times. 10.sup.-5 1.2 0.3 97.8 0.3
Example 2
Topical Formulations
[0043] Table 2 shows twelve exemplary lyophilized formulations that
were prepared according to the invention. Each formulation was
prepared by adding the respective components in the indicated
amounts to a standard 2 ml lyophilization vial. The column heading
"Toxin (ng/vial)" refers to the amount of botulinum toxin type A
neurotoxin present (in nanograms per vial), while the column
heading "peptide (mg/ml)" refers to the amount of the peptide
RKKRRQRRR-Q-(K).sub.15-Q-RKKRRQRRR present (in milligrams per
vial). Table 2-1 shows the same twelve exemplary lyophilized
topical botulinum toxin formulations as in Table 2, except that the
numerical entries in Table 2-1 refer to the respective weight
percent of the components relative to the total weight of the
lyophilized formulation.
TABLE-US-00003 TABLE 2 Exemplary Botulinum Toxin Formulations
Histidine hydrochlo- Boric Propyl Polysorbate Formu- Toxin Peptide
Histidine ride Sucrose Trehalose Sorbitol Glycerol Acid Gallate 20
lation (ng/vial) (mg/vial) (mg/vial) (mg/vial) (mg/vial) (mg/vial)
(mg/vial) (mg/vial) (mg/vial) (mg/vial) (mg/vial) 36 11 0.015 0.45
0.10 36.0 0.10 37 11 0.015 0.45 0.10 33.0 3.0 0.10 38 11 0.015 0.45
0.10 33.0 3.0 0.10 0.10 39 11 0.015 0.45 0.10 34.0 2.0 0.10 40 11
0.015 0.45 0.10 36.0 0.10 41 11 0.015 0.45 0.10 33.0 3.0 0.10 42 11
0.015 0.45 0.10 35.0 1.0 0.10 43 11 0.015 0.45 0.10 34.0 2.0 0.10
44 11 0.015 0.45 0.10 34.0 2.0 0.10 0.10 45 1.1 0.015 0.45 0.10
36.0 0.10 46 1.1 0.015 0.45 0.10 36.0 0.10 47 1.1 0.45 0.10 36.0
0.10
TABLE-US-00004 TABLE 2-1 Exemplary Botulinum Toxin Formulations
(amounts expressed in terms of weight percent relative to total
weight of the lyophilized formulation) Toxin Histidine Boric Propyl
Formulation (.times.10.sup.-5) Peptide Histidine hydrochloride
Sucrose Trehalose Sorbitol Glycerol Acid Gallate Polysorbate 36
3.00 0.041 1.23 0.27 98.2 0.27 37 3.00 0.041 1.23 0.27 90.0 8.2
0.27 38 3.00 0.041 1.22 0.27 89.8 8.2 0.27 0.27 39 3.00 0.041 1.23
0.27 92.7 5.5 0.27 40 3.00 0.041 1.23 0.27 98.2 0.27 41 3.00 0.041
1.23 0.27 90.0 8.2 0.27 42 3.00 0.041 1.23 0.27 95.5 2.7 0.27 43
3.00 0.041 1.23 0.27 92.7 5.5 0.27 44 3.00 0.041 1.22 0.27 92.5 5.4
0.27 0.27 45 3.00 0.041 1.23 0.27 98.2 0.27 46 3.00 0.041 1.23 0.27
98.2 0.27 47 3.00 1.23 0.27 98.2 0.27
Example 3
Reconstitution of Formulations
[0044] The lyophilized formulations listed in Tables 1 and 2
generally are used after reconstitution with a diluent. Generally,
any pharmaceutically acceptable diluent that does not undergo
undesirable reactions with the components contained in the
respective formulation may be used. For example, the formulations
listed in Tables 1 and 2 may be reconstituted using water, saline,
or phosphate buffered saline. Optionally, one or more additives may
be included in the diluent to control or improve certain properties
of the diluent, non-limiting examples of which include viscosity
enhancers (e.g., a poloxamer, such as poloxamer 188 or 407),
anti-oxidants (e.g., BHT or methionine), co-solvents (e.g., an
alcohol, such as ethanol), and/or tonicity adjusters (e.g., a salt,
such as sodium chloride).
[0045] For the formulations listed in Tables 1 and 2, it is
convenient to reconstitute using 1 ml of diluent, since the
formulations are stored in 2 ml vials. Thus, the invention
expressly contemplates reconstitution of each formulation listed in
Tables 1 and 2 with 1 ml of diluent. Two exemplary diluents that
are useful with the formulations of the invention is described
below in Tables 3 and 3-1. It is to be understood that the
reconstituted formulations of Table 1 recited in Examples 5 and 6
below were reconstituted using the diluent set forth in Table 3,
unless otherwise indicated.
TABLE-US-00005 TABLE 3 Exemplary Diluent for Reconstituting
Formulations Amount needed to Reconstitute one 2 ml vial of
Component Function Drug Product Poloxamer 407 Viscosity Enhancer
170 mg Butylated Anti-oxidant 0.1 mg hydroxytoluene Ethanol
Co-solvent 0.725 mg Sodium Chloride Tonicity adjuster 7.53 mg Water
Solvent 852.5 mg
TABLE-US-00006 TABLE 3-1 Additional Exemplary Diluent for
Reconstituting Formulations Amount needed to Reconstitute one 2 ml
vial of Component Function Drug Product Poloxamer 407 Viscosity
Enhancer 170 mg Sodium Chloride Tonicity adjuster 7.53 mg Water
Solvent 852.5 mg
Example 4
Topical Formulations
[0046] In certain preferred embodiments, the topical formulations
of the invention contain botulinum neurotoxin, a carrier peptide, a
buffer, a sugar, and a non-ionic surfactant. For instance, certain
useful topical formulations may contain 4.1.times.10.sup.-6-0.22
wt. % botulinum neurotoxin type A, 2.5.times.10.sup.-5-0.1 wt. % of
the peptide RKKRRQRRR-Q-(K).sub.15-Q-RKKRRQRRR, 1-1.5 wt. %
histidine, 0.1-0.5 wt. % histidine hydrochloride, 97-99 wt. %
sucrose, and 0.1-0.3 wt. % polysorbate 20. If desired, such
formulations may be reconstituted using the diluent set forth in
Table 3.
Example 5
Treatment of Allergic Rhinitis
Materials and Methods
Reagents
[0047] The following reagents were used: Formulation 22 reported in
Table 1 above (reconstituted with the diluent reported in Table 3),
a control gel (Revance Therapeutics, Inc., Newark, Calif.),
ovalbumin, aluminum hydroxide, gelatin, sodium phosphate,
diaminobenzadine (Sigma-Aldrich, St. Louis, Mo.), sheep anti-VIP
antibody (Millipore, Billerica, Mass.), biotinylated rabbit
anti-sheep antibody and ABC kit (Vector Labs, Burlingame, Calif.),
Botulinum toxin type A complex stock solution (Metabiologics Inc.,
Madison, Wis.).
Animals
[0048] Female Sprague-Dawley rats weighing 200-250 g (Charles River
Laboratories Inc, Hollister, Calif.) were used in the allergic
rhinitis model. Guinea pigs weighing 283-325 g (Charles River,
Raleigh, N.C.) were used in the comparative safety study. Animals
were housed in a vivarium with a 12 h light/dark cycle and a
controlled temperature. Food and water were provided ad libitum.
All procedures in this study were performed in accordance with the
guidelines detailed in the Guide for Care and Use of Laboratory
Animals published by the National Academy of Sciences and approved
by the Institutional Animal Care and Use Committee.
Ovalbumin-Induced Allergic Rhinitis Model
[0049] The allergy induction protocol consisted of a series of
seven intraperitoneal injections of ovalbumin (0.3 mg) and aluminum
hydroxide powder (30 mg) suspension in 0.9% saline (1 mL)
administered every other day under anesthesia (-2% isoflurane in
O.sub.2). Ovalbumin (2 mg) in 0.9% saline (20 .mu.L) was then
intranasally instilled daily for a total of seven doses under
anesthesia (.about.2% isoflurane in O.sub.2).
[0050] Sneezing and nasal itching (indicated by nasal rubbing) are
useful indications of allergic rhinitis in rats and represent two
of the four traditional clinical symptoms (along with rhinorrhea
and congestion) monitored in patients. Consequently, a performance
severity assessment (PSA) scale was established to score the extent
of these two nasal allergic signs following antigen challenge.
Clinical signs were scored prior to induction at baseline,
following induction to establish maximal allergic signs on day 0
and on days 3, 5 and 7 following treatment using numerical scores:
a) itching (rubbing nose): 0, none; 1, <30 times; 2, 30-50
times; 3, .gtoreq.50 times and b) sneezing: 0, none; 1, <3
times; 2, 3-10 times; 3, .gtoreq.10 times (30 minute period). The
sum of scores for itching and sneezing comprised the composite PSA
score.
[0051] After completing the induction process, animals which did
not respond were excluded from the study. The responding animals
were randomly divided into two groups (n=7 per group) and treated
with either reconstituted Formulation 22 (0.4 ng dose per rat,
equivalent to 100 U of botulinum toxin) or control diluent (total
volume of 40 .mu.L per rat). Administration of reconstituted
Formulation 22 and the control diluent was achieved by inserting a
pipette loaded with the appropriate composition into the nasal
cavity of the test animals and expelling the composition therein.
Administration of intranasal ovalbumin was continued every other
day to maintain allergic signs. The animals were evaluated prior to
reconstituted Formulation 22 (or control) treatment and
post-treatment on days 3, 5 and 7 consistent with the emergence of
treatment effect previously reported on day 5 after Botulinum
neurotoxin type A treatment.
Histological and Immunohistochemical Analysis
[0052] Animals were euthanized on day 10 after treatment with
reconstituted Formulation 22 or control diluent. The nasal tissues
were harvested and fixed in 10% formalin overnight, routinely
processed and transversely cut into 5 .mu.m sections at a depth of
1.5 mm from the nostril. Alternating sections were processed for
standard hematoxylin and eosin (H&E) staining and vasoactive
intestinal peptide (VIP) immunohistochemical staining The sections
for VIP staining were incubated overnight at 4.degree. C. with the
sheep anti-VIP antibody (1:1000 dilution). Sections were washed
followed by incubation with biotinylated rabbit anti-sheep (1:200
dilution) for 30 minutes at room temperature. VIP immunoreactivity
was then visualized using an ABC kit (1:100 dilution) with
diaminobenzadine as chromogen.
Safety Testing in Guinea Pigs
[0053] Guinea pigs were randomly assigned (three per group) to each
dose group with dose levels selected based on previous dose ranging
studies. Formulations 27-29 were reconstituted using the diluent
described in Example 3 and then administered, one formulation per
animal, to each three-animal dose group. Administration was
achieved by using a loaded pipette as described above. Following
intranasal administration, all animals were observed and weighed
daily for the duration of the study. The No Adverse Effect Level
(NOAEL) was established as the highest dose causing no adverse
clinical observation and no significant difference in mean body
weight and overall body weight change compared to controls by
t-test. The LD50 was calculated using nominal logistic platform in
JMP.RTM. 9.0. (SAS Institute Inc.).
Data Analysis
[0054] One-way ANOVA was used to assess statistical significance
between PSA scores across groups and days of observation. Post-hoc
Scheffe tests were performed to assess significance between
specific day pairs of observation in the treated animal group.
Student's t-test was used for comparison of treatments within
treatment day and for comparison of body weights across treatment
groups. For statistical significance, a confidence level of
p<0.05 was used.
Results
Effect of Topical Formulations on Allergic Rhinitis in Rats
[0055] Following the initial ovalbumin challenge, both itching and
sneezing incidence increased significantly. The PSA score
(mean.+-.SEM) increased from 1.0.+-.0.2 to 3.7.+-.0.4 in the
treated group (p<0.01), and from 1.1.+-.0.1 to 4.3.+-.0.4 in the
control group (p<0.01). There were no significant differences
between the two groups either pre-challenge or post-challenge prior
to treatment (p>0.05).
[0056] Allergic rhinitis severity was assessed on day 3, 5 and 7
after treatment with reconstituted Formulations 22 or the control
treatment. The PSA score did not significantly change in the
control group, while the PSA score progressively decreased in the
reconstituted Formulation 22 group (FIG. 1). One-way ANOVA analysis
indicated that the PSA score changes were significantly different
between the reconstituted Formulation 22 and control groups
(F=7.277, p=0.009). Subsequent post-hoc Scheffe tests showed that
the PSA scores were significantly reduced on days 3, 5 and 7 as
compared to pre-treatment in the reconstituted Formulation 22 group
(p<0.05, p<0.01, p<0.01, on days 3, 5 and 7,
respectively). As compared to the pre-challenge baseline, the PSA
score in the reconstituted Formulation 22 group was still
significantly elevated on day 3 (p<0.05), but the scores on days
5 and 7 did not significantly (p>0.05) differ from the normal
baseline score indicating that the allergic symptoms were
essentially resolved to the normal level by day 5 following
reconstituted Formulation 22 treatment. PSA scores for
reconstituted Formulation 22 treatment were significantly reduced
compared to same day control animals (p<0.05). PSA scores for
control animals remained significantly elevated (p<0.01) from
normal baseline throughout the experiment indicating the sustained
induction of allergic rhinitis clinical signs in the model
throughout the course of the experiment.
Histological and Immunohistochemical Evaluation of Topical
Formulation Treatment Effects
[0057] The H&E stained sections of control animals revealed
typical signs of inflammatory pathology including edema, congestion
and vascular dilatation in nasal mucosa across the cavity,
particularly in turbinate (FIG. 2b) and lateral nasal wall (FIG.
2e), as compared to normal control animals (FIGS. 2a and d).
Reconstituted Formulation 22 treatment resulted in essentially
complete resolution of inflammatory findings (FIG. 2).
Additionally, hyperplasia of serous glands also was found in some
control animals (FIG. 2b). The nasal mucosa of
reconstituted-Formulation 22-treated animals (FIGS. 2c and f)
appeared essentially normal with only mild congestion (i.e. on
lateral nasal wall of this animal specimen shown in FIG. 2e) on day
10 after treatment. No signs of atrophy or degeneration of serous
glands were found after the reconstituted Formulation 22
treatment.
[0058] VIP expression was dramatically increased following
ovalbumin challenge and observed in the control animals (FIG. 3b)
in contrast to the normal animals (FIG. 3a), especially around
blood vessels and serous glands. Following reconstituted
Formulation 22 treatment, VIP expression in the nasal mucosa
decreased markedly (FIG. 3c) and appeared comparable to normal
animal tissue samples (FIG. 3a).
Safety Evaluation of Reconstituted Formulations Compared to
Botulinum Neurotoxin Type Complex in Guinea Pigs
[0059] The safety profile of reconstituted Formulations 27-29
following intranasal dosing was compared to botulinum type A
complex in guinea pigs which are highly sensitive to the toxic
effects of botulinum type A complex, thus providing a conservative
estimate of safety for reconstituted Formulation 27-29 (FIG. 4).
Death was observed in groups of animals treated with 110000 U of
reconstituted Formulation 29, and 1800 U and 3600 U of botulinum
type A complex. The LD50 for reconstituted Formulations 27-29 and
botulinum type A complex via intranasal route of administration was
determined as 108350 U and 1836 U, respectively. Dose levels of
reconstituted Formulation 28 up to 55000 U/animal were well
tolerated with no abnormal clinical observations and no significant
difference in mean daily body weight or overall body weight gain as
compared to control (p=0.2278 body weight gain). There was no
significant difference in mean daily body weight or overall body
weight gain of animals treated with botulinum type A complex at 900
U (p=0.1963 for body weight gain) compared to control. The No
Adverse Effect Level (NOAEL) observed for intranasal dosing of
guinea pigs was 27500 U/animal for reconstituted Formulation 27 and
900 U/animal for botulinum type A complex, indicating reconstituted
Formulations 27-29 are approximately 31-fold safer compared to
botulinum type A complex via the intranasal route of
administration. Further, this NOAEL dose in guinea pigs is
approximately 250-fold higher than the dose shown to be effective
in treating allergic rhinitis in the rat model.
Discussion
[0060] The PSA scale was shown to be sensitive and specific in
tracking the onset of clinical signs of rhinitis. The typical total
nasal symptom score used in clinical studies of rhinitis treatments
tracks sneezing, itching, rhinorrhea and congestion. For this study
clinical observation of animals permitted quantitation of sneezing
and itching (nasal rubbing) whereas congestion was addressed
qualitatively as part of the histopathology assessment Rhinorrhea
was difficult to quantitate in rats and thus not included, however,
it generally tracks with the other symptoms when evaluated in
clinical treatment of rhinitis. Treatment with reconstituted
Formulation 22 but not control resulted in significant reduction in
PSA score by day 3 following treatment as compared to control and
with essentially full resolution to normal baseline levels by day
5. In accordance with previous studies that treated rhinitis by
injecting botulinum type A complexes in animals and in humans,
these results indicate that the topical intranasal application of
reconstituted Formulation 22 also can relieve clinical signs in a
rat model of allergic rhinitis.
[0061] Histological staining of nasal tissues of allergic animals
revealed significant degrees of mucosal edema, congestion and
vascular dilatation along with hyperplasia of serous glands which
were resolved to essentially normal baseline levels following
reconstituted Formulation 22 treatment. This effect of
reconstituted Formulation 22 treatment on the inflammatory response
associated with allergic rhinitis was further characterized by
showing that the tissue level of VIP, a known mediator of nasal
glandular secretions and the inflammatory process, was reduced
essentially to normal levels following reconstituted Formulation 22
treatment.
[0062] Allergic rhinitis is caused by the interaction of allergens
with inflammatory cells, resulting in release of vasoactive and
proinflammatory mediators within the nasal mucosa. Regulatory
peptides, such as VIP, may play an important role in the
hypersecretion of allergic rhinitis where a high density of VIP
expression has been shown in nasal mucosa in allergic rhinitis
patients and rats. It is therefore noteworthy that reconstituted
Formulation 22 treatment reduced VIP expression to baseline levels
and the suppression of VIP activity is associated with the
reduction of congestion, vascular dilatation and edema.
[0063] The current study shows that Formulation 22, reconstituted
as described above, provides efficient transmucosal penetration in
this rat model, eliminating the need for treatment by injection or
nasal sponge packing, as reported in previous studies. Transmucosal
flux of botulinum neurotoxin type A and the gel nature of
reconstituted Formulation 22 helps to localize the applied dose to
the intended treatment site, thereby limiting spread of liquid
botulinum type A to the gut via nasopharyngeal drainage and
potential systemic toxicity. This safety profile of reconstituted
Formulations 27-29 were confirmed in the comparative safety study
with botulinum type A complex in guinea pigs showing reconstituted
Formulations 27-29 to be approximately 31-fold safer compared to
botulinum type A complex. The No Adverse Effect Level (NOAEL) dose
in guinea pigs is approximately 250-fold higher than the dose shown
to be effective in treating allergic rhinitis in the rat model thus
providing a large therapeutic window in preclinical models.
Example 6
Safety Profile of Purified Botulinum Neurotoxin Type A
Materials and Methods
Reagents
[0064] Reconstituted Formulation 25 as described above was
manufactured at Revance Therapeutics, Inc. (Newark, Calif.), and
botulinum type A complex was purchased from Metabiologics (Madison,
Wis.) as a 1 mg/mL solution. Simulated gastric fluid (SGF) and
simulated intestinal fluid (SIF) were prepared per United States
Pharmacopeia. In some cases, the SGF was used without addition of
pepsin to study the contribution of the enzyme.
Animals
[0065] Male rats, weighing 200-250 g, were purchased from Charles
River Laboratories (Hollister, Calif.). The use of animals in this
study was approved by the local Institutional Animal Care and Use
Committee (IACUC). Rats were housed in a vivarium with a 12 h
light/dark cycle and controlled temperature. Food and water were
provided ad libitum.
Oral Toxicity
[0066] A single dose 14-day oral toxicity study in rats was
conducted comparing oral toxicity of reconstituted Formulations
30-35 and botulinum type A complex. Animals (fed state) were dosed
via oral gavage at 4 mL/kg. For reconstituted Formulations 30-35,
rats were dosed with 1.03.times.10.sup.7 up to 1.76.times.10.sup.8
U/kg of toxin using a pipette as described in Example 1. For
botulinum type A complex dose group, rats were dosed with
1.31.times.10.sup.5 up to 4.20.times.10.sup.6 U/kg. These dose
ranges were based on preliminary dose ranging studies (data not
shown). All animals were observed and weighed daily throughout the
course of the study. The NOAEL was established as the highest dose
causing no adverse clinical observation and no significant
difference in mean body weight and overall body weight change
compared to controls by t-test (Excel 2003, Microsoft, Washington,
USA). The LD.sub.50 was calculated using the nominal logistic
platform in JMP.RTM. 9.0. (SAS Institute Inc., NC, USA).
In Vitro Assays in Gastrointestinal Model
[0067] In order to study the gastric effect on toxin stability,
reconstituted Formulation 25 and botulinum type A complex were
incubated in SGF at 9000 U/mL at 37.degree. C. The potency of
reconstituted Formulation 25 and botulinum type A complex were
measured at T=0 and at each subsequent time point. Due to the
differences in stability profile of reconstituted Formulation 25
and botulinum type A complex, reconstituted Formulation 25 and
botulinum type A complex were incubated in SGF using adjusted time
courses. In the case of reconstituted Formulation 25, the toxin was
exposed to SGF for less than one minute before diluted for potency
testing due to rapid inactivation. In the case of botulinum type A
complex, potencies were tested on samples taken at 5 minute, 15
minutes, 30 minutes, and 60 minutes.
Proteolysis Effects
[0068] In order to study the enzymes effects on toxin stability,
reconstituted Formulation 25 and botulinum type A complex were
incubated in SGF at 9000 U/mL at 37.degree. C. with pepsin. Again,
the potency of reconstituted Formulation 25 and botulinum type A
complex were measured at T=0 and at each subsequent time point; in
the case of reconstituted Formulation 25, the toxin was exposed to
SGF for less than one minute before dilution for its potency test.
In the case of botulinum type A complex, samples were taken at
5.degree. minute, 15.degree. minutes, 30.degree. minutes, 1.degree.
hour, and 2.degree. hours for its potency test.
[0069] In order to study the enzymes effect on toxin stability in
SIF, reconstituted Formulation 25 and botulinum type A complex were
incubated in SIF for up to 2 hours. Samples taken at 0, 30 minutes,
and 2 hours were tested for potency.
[0070] In order to mimic the normal physiological pattern where the
toxin is first exposed to gastric fluid and then exposed to
intestinal fluid, a separate experiment was conducted wherein
reconstituted Formulation 25 and botulinum type A complex were
first incubated in SGF for 30 minutes, then transferred to SIF and
further incubated for up to 2 hours. Samples taken at 0, 30
minutes, and 2 hours were tested for their potency. Samples from
each test condition were tested in mouse potency assay via
intraperitoneal (IP) injection for lethality at 72 hr. Dose
response data were analyzed using the nominal logistic platform in
JMP.RTM. 9.0. (SAS Institute Inc.) to derive an LD50 value and to
calculate relative potency compared to a reference standard
material.
[0071] Data Analysis.
[0072] For each time course study, the potency at T=0 was measured.
The potency at each subsequent time point was expressed as the
percentage of T=0,
% Potency=T.sub.x minutes/T.sub.0.times.100%
[0073] In botulinum type A complex stability study in SGF, the
logarithmic curve fits were formatted using Microsoft Office Excel
2003 (Microsoft, Redmond, Wash., USA) to compute the half-life of
BoNTA complex stability. The half-life was then calculated.
Results
Oral Toxicity
[0074] To further characterize the safety profile of reconstituted
Formulations 30-35 following oral dosing and to compare
reconstituted Formulations 30-35 with conventional botulinum type A
complex, rats were dosed orally via gavage with escalating doses to
determine oral LD50 and NOAEL for the two toxin formulations. Death
was observed in groups of animals treated with higher than
2.06.times.10.sup.7 U/kg of reconstituted Formulation 31, and
2.63.times.10.sup.5 U/kg of botulinum type A complex (Table 4). The
oral LD50 for reconstituted Formulations 30-35 and botulinum type A
complex was determined to be 1.19.times.10.sup.8 U/kg and
5.03.times.10.sup.6 U/kg, respectively.
TABLE-US-00007 TABLE 4 Dose response Dose in LD50 (#death/total #
Test article U (U/kg) Formulation tested) Topical 1.76E+08 35 1/3
Formulation 8.25E+07 34 3/6 5.87E+07 33 0/3 4.13E+07 32 1/6
2.06E+07 31 2/6 1.03E+07 30 0/6 botulinum type 4.20E+06 1/3 A
complex 1.40E+06 1/3 5.25E+05 1/6 4.66E+05 0/3 2.63E+05 1/12
1.31E+05 0/6
[0075] Animals dosed with toxin concentrations of
1.03.times.10.sup.7 U/kg of reconstituted Formulation 30 or
1.31.times.10.sup.5 U/kg of botulinum type A complex showed no
abnormal clinical observations and no significant difference in
mean daily body weight or overall body weight gain as compared to
control (p=0.4952 and 0.4163 for body weight gain reconstituted
Formulation 30 and botulinum type A complex, respectively),
indicating reconstituted Formulation 30 is approximately 80-fold
less toxic compared to botulinum type A complex via the oral route
of administration on a unit-for-unit basis.
In vitro Assays
[0076] To further characterize the distinct oral safety profiles of
botulinum type A complex and reconstituted Formulation 25 observed
in vivo, comparison was made using SGF and SIF model systems.
Incubation of reconstituted Formulation 25 in SGF resulted in
complete loss of detectable activity within one minute of
incubation consistent with a half-life of less than 6 seconds
(lower limit of detection for the potency assay is 0.1% of starting
activity, i.e., ten half-lives of decay). In contrast, botulinum
type A complex slowly lost its activity with a half-life of 9
minutes.
[0077] Besides pH extremes, the other stresses that the botulinum
neurotoxin type A must endure come from proteases, such as pepsin
in the gastric fluid. There was no detectable difference in
reconstituted Formulation 25 stability in SGF in the presence of
pepsin; interestingly, the stability of botulinum type A complex in
SGF is enhanced in the presence of pepsin with a half-life of
nearly 17 minutes, almost double the half-life in the absence of
pepsin and at least 170-fold greater than reconstituted Formulation
25.
[0078] When incubated in SIF, both botulinum type A formulations
were susceptible to protease digestion where reconstituted
Formulation 25 was degraded faster than botulinum type A complex,
indicating the non-toxin accessory proteins protect the toxin from
enzyme digestion (Table 5). However, incubation of the botulinum
type A complex in SGF (pepsin at pH 1.2) prior to exposure of the
complex to SIF actually enhanced the stability of botulinum type A
complex in SIF (Table 6) exhibiting a half-life approximately 30
minutes. Thus, under conditions that simulate the path of the
botulinum type A complex through the gastrointestinal tract (i.e.,
gastric exposure followed by intestinal exposure), the nontoxic
components of the complex protected botulinum neurotoxin from
proteolysis and actually enhanced subsequent stability in SIF
whereas reconstituted Formulation 25 was very rapidly inactivated
in SGF and did not exhibit any detectable activity in the
intestinal environment. When reconstituted Formulation 25 was first
incubated in SGF and then in SIF, no toxin activity was observed,
indicating the SGF caused changes in the toxin protein which were
not recovered in SIF.
TABLE-US-00008 TABLE 5 Time (minutes) 0 30 120 reconstituted
Formulation 25 100.0% 0% 0% botulinum type A complex 100.0% 4.1%
0.7% botulinum type A complex 100% 46.5% 3.6% (pre-incubation in
SGF)
TABLE-US-00009 TABLE 6 Half-life (min) botulinum type reconstituted
Condition A complex Formulation 25 SIF <2 <0.1 Pre-incubation
in SGF 26 <0.1
Discussion
[0079] The differences in reported safety margin of 150 kDa to
botulinum type A complex may due to the differences in toxin
preparations/formulation and the methods used to detect toxin
biological activity. In this study, the oral toxicity of botulinum
type A complex was significantly greater than that observed for
purified 150 kDa neurotoxin. The oral LD.sub.50 values for
reconstituted Formulations 30-35 and botulinum type A complex were
determined to be 1.19.times.10.sup.8 U/kg and 5.03.times.10.sup.6
U/kg, respectively; the NOAEL for reconstituted Formulation 30 and
botulinum type A complex were 1.03.times.10.sup.7 U/kg and
1.31.times.10.sup.5 U/kg, respectively, indicating reconstituted
Formulation 30 was approximately 80-fold less toxic compared to
botulinum type A complex via the oral route of administration.
Reconstituted Formulation 25 bioactivity was undetectable within
one minute in SGF with a half-life of less than 6 seconds whereas
the half-life of the BoNTA complex was approximately 17 minutes in
SGF. The intestinal stability of botulinum type A complex SIF was
enhanced by >10 fold if the toxin was pre-incubated in SGF,
whereas when reconstituted Formulation 25 was first incubated in
SGF and then in SIF, no toxin activity was observed, indicating the
gastric fluid caused irreversible loss of activity. These findings
in vitro may underlie the mechanism by which reconstituted
Formulation 30, when dosed orally in rats, is approximately 80
times less toxic than botulinum type A complex due to its
instability in the gastric and intestinal fluids, the natural path
of neurotoxin through gastrointestinal tract when ingested.
Example 7
Phase 2 Clinical Trial to Evaluate Safety and Efficacy of Botulinum
Toxin Type A Topical Gel in Treating Allergic Rhinitis
Design and Methodology
[0080] A double-blind, randomized, placebo controlled study was
conducted with 51 subjects enrolled. Subjects were enrolled in one
of two (2) parallel treatment groups with 26 subjects in the test
article group and 25 subjects in the control group, following
satisfaction of entry criteria and screening procedures. Each
subject received either a test article or placebo.
Subjects
[0081] Subjects participating in the study met the following
eligibility criteria: [0082] predominately seasonal allergic
rhinitis with a history of seasonal allergic rhinitis, for at least
12 months, having required intervention/medication and confirmed by
documented medical history; [0083] confirmed as reactive to rye
grass seasonal allergen on skin prick testing (greater than 3 mm)
or positive blood specific IgE testing rye grass allergen, within
the prior 12 months; [0084] total nasal symptom score of at least 7
(using a composite scale of 0-12 based on 0-3 scales for each of
four symptoms) averaged from screening to treatment (day 0); [0085]
outpatient, male or non-pregnant, non-nursing females, 18-65 years
of age, in good general health; [0086] female subjects of
childbearing potential having a negative urine pregnancy test
result at baseline and practicing a reliable method of
contraception throughout the study; and [0087] willing to refrain
from use of medications for rhinitis symptoms (such as
decongestants, corticosteroids--oral, inhaled and nasal sprays) for
1 week prior and 2 weeks post treatment.
Test Article/Placebo, Dose, and Mode of Administration
[0088] The test article in this Example was a topical gel
containing "Formulation 24", as listed above in Table 1.
Formulation 24 was used to prepare the topical gel formulation,
which was composed of 25 ng/vial purified and lyophilized 150 kDa
botulinum toxin type A and approximately 9 mcg/mL of the absorption
enhancing peptide excipient, reconstituted with 1.0 mL of a
poloxamer diluent, along with inactive ingredients, giving a
concentration of 25 ng/mL or 5 ng/0.2 mL. The peptide excipient
used was the antimicrobial peptide having amino acid sequence,
Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-(Lys).sub.15-Gly-Arg-Lys-Lys-Arg--
Arg-Gln-Arg-Arg-Arg. The poloxamer diluent used was 16.5% poloxamer
407 and 0.9% sodium chloride in water, with BHT and ethanol (see
Table 3). Other inactive ingredients included histidine, histidine
hydrochloride, sucrose, and polysorbate 20, in the amounts provided
above in Table 1.
[0089] Approximately 0.2 mL total volume (5 ng) of the test article
was used as the applied test dose, using topical administration.
(The peptide enables the delivery of botulinum toxin to the
underlying tissue.) The placebo comprised a topical gel formulation
of only the inactive ingredients of Formulation 24 (histidine,
histidine hydrochloride, sucrose, and polysorbate 20, in the
amounts provided above in Table 1), reconstituted with the
poloxamer diluent. Again approximately 0.2 mL total volume was used
as the applied placebo dose, using topical administration.
Accordingly, a dose of 5 ng of the test article was compared to
placebo.
[0090] The topical gels were prepared by an unblinded, trained
study staff member. Formulation 24 and control were supplied in a
professional, single-use reconstitution/administration apparatus,
containing a vial of lyophilized botulinum toxin Type A, as the
active pharmaceutical ingredient (25 ng/mL or placebo), formulated
with peptide excipient (9 mcg/mL), and other inactive ingredients.
The apparatus also contained a cartridge of poloxamer diluent for
reconstitution. The placebo control was a lyophilized vial of
inactive ingredients. The reconstituted formulations provided the
gels for topical application.
[0091] For each subject, 0.5 mL of gel containing either
Formulation 24 or placebo was divided between four swabs, with two
swabs for each of the two nostrils, for application to the inferior
turbinates and adjoining intranasal mucosal surfaces using a nasal
speculum. Some of the product was absorbed by the swab, and left on
the swab. Thus, while 0.5 mL of gel was used to wet swabs, this
lead to a transfer of approximately 0.2 mL to the target dose site.
Specifically, swab recovery studies based on mass transfer showed
that when one loads gel (containing Formulation 24 or placebo) onto
a swab, where approximately 0.117 g of gel was loaded on a swab,
approximately 0.083 g of gel was recovered on the swab after
application in accordance with study protocol, indicating that
approximately 0.034 g of gel was applied intranasally. It follows
then that using a total of four swabs (two per nostril), the total
amount of gel transferred was approximately 0.034 g.times.4, which
equals approximately 0.136 g of gel. As the density of the gel was
1.027 g/mL, approximately 0.132 mL of gel was calculated to have
been applied intranasally to each subject. This value was rounded
up to 0.2 mL, as a conservative estimate for safety in the clinical
trial.
[0092] Subjects received a single intranasal application of either
the test article (containing Formulation 24) or a matched placebo
gel on day 0. The gel was allowed to remain on for 30 minutes and
then removed by saline flush. Subjects were monitored for safety
throughout the study, at day 2 and weeks 2, 4, and 8, and for local
irritation of treatment areas at baseline and post-treatment day 0
and day 2.
Efficacy Assessments
[0093] Efficacy was assessed by total nasal symptom (TNS) score,
Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ), peak
nasal inspiratory flow (PNIF), and peak expiratory flow (PEF) with
forced expiratory volume (FEV1), all recorded by the subject and
reported at baseline and weeks 2, 4, and 8.
[0094] TNS-4 score is the sum of individual scores for rhinorrhea,
nasal congestion, nasal itching, and sneezing, each measured on an
ordinal scale of 0, 1, 2 or 3, representing no symptoms, mild,
moderate, or severe symptoms, respectively. The individual
components of the TNS score are recorded by the subject daily and
reported at follow-up visits (pre- and post-allergen challenge
where applicable).
[0095] RQLQ is a questionnaire measuring the functional problems
(physical, emotional, social, and occupational) most troublesome to
adults with either seasonal or perennial of allergic or
non-allergic rhinoconjunctivitis (see, Juniper E F, et al.
Development and testing of a new measure of health status for
clinical trials in rhinoconjunctivitis. Clin Exper Allergy 1991;
21: 77-83., 1991, which is hereby incorporated by reference in its
entirety). The RQLQ includes 28 questions in 7 domains (activity
limitation, sleep problems, nose symptoms, eye symptoms,
non-nose/eye symptoms, practical problems, and emotional function)
of which 3 are `patient-specific` questions in the activity domain,
allowing patients to select 3 activities where there are most
limited by their rhinoconjunctivitis. Patients recall how bothered
they were by their rhinoconjunctivitis during the previous week and
respond to each question on a 7-point scale (0=not
affected-6=extremely affected). The overall RQLQ score is the mean
of all 28 responses and the individual domain scores are the means
of the items in those domains. The RQLQ has been validated and used
extensively throughout the world in clinical practice and clinical
trials (see Juniper E F, et al. Validation of a standardised
version of the Rhinoconjunctivitis Quality of Life Questionnaire. J
Allergy Clin Immunol 1999; 104: 364-9).
[0096] As an additional efficacy assessment, allergen challenge was
performed at baseline and week 4, wherein the subject was exposed
to cognate allergen (subject confirmed as reactive by prior skin
test; allergen extract applied to nasal septum on paper filter
disk), and nasal symptoms and PNIF were recorded
post-challenge.
[0097] A quantitative airflow measure also was employed.
Specifically, due to the high comorbidity of bronchial reactivity
(which would change both mouth and nasal airflow), the measure of
nasal resistance to flow was measured as a pure rhinitis
quantitative measure. This represents the difference between nasal
and mouth air flow. The smaller the gap, the less nasal resistance
was present.
Efficacy Evaluations
[0098] Efficacy assessments were performed at baseline and weeks 2,
4, and 8 by: [0099] total nasal symptom score: combination of
rhinorrhea, nasal congestion, nasal itching, and sneezing, recorded
daily by each subject and reported at visits (pre- and
post-allergen challenge where applicable); [0100] individual nasal
symptom scores for rhinorrhea, nasal congestion, nasal itching and
sneezing, recorded daily by subject and reported at visits and
scored at office visit (pre- and post-allergen challenge where
applicable); [0101] rhinoconjunctivitis quality of life
questionnaire (RQLQ) completed weekly by subjects; and [0102] peak
nasal inspiratory flow (PNIF) with peak expiratory flow (PEF) by
the subject daily and at visits (pre- and post-allergen challenge
where applicable).
Safety Assessments
[0103] Clinical Laboratory Data: As outlined in Table 7,
non-fasting samples for chemistry were collected at screening and
week 8 (or early discontinuation).
TABLE-US-00010 TABLE 7 Clinical Laboratory Tests Serum Chemistry
Hematology Urinalysis Additional Tests Glucose Hemoglobin Specific
gravity Urine Pregnancy Total bilirubin Hematocrit pH (WOCBP only)
Alanine aminotransferase Total and differential Glucose Serum
pregnancy Aspartate aminotransferase leukocyte count Protein test
at week 8 Alkaline phosphatase Red blood cell count Blood (or early
Blood Urea Nitrogen Platelet count Bilirubin discontinuation)
Ketones if UPT is positive
[0104] Pregnancy Testing: All women of childbearing potential had a
urine pregnancy test at screening and treatment (day 0) and if
either result was positive, the subject was not allowed to
participate in the study. Women of childbearing potential also had
a urine pregnancy test at their final study visit (week 8 or early
discontinuation). If the test was positive, the pregnancy was
confirmed by a serum pregnancy test.
[0105] Local Irritation Assessment: All application sites were
evaluated for any irritation, pre-treatment and post-treatment,
using the Erythema Assessment (Table 8) and the Clinical
Signs/Symptoms Descriptors (Table 9).
TABLE-US-00011 TABLE 8 Erythema Assessment Rating Description 0 No
reaction/No erythema 1 Minimal erythema (barely perceptible) 2
Strong erythema (easily visible) 3 Strong erythema, spreading
outside of treated site 4 Strong erythema, spreading outside of
treated site with either edema (swelling) or vesicles (elevated,
circumscribed lesions up to 1 cm in size that are filled with
serous fluid) 5 Severe reaction with ulceration (irregularly sized
and shaped erosions)
TABLE-US-00012 TABLE 9 Clinical Signs/Symptom Descriptors
Descriptor Description O No Clinical Signs/Symptoms E Edema
(swelling) S Scaling (shedding of dead cells) F Fissures (linear
breaks in the mucosa) C Crusts (colored masses of exudates) V
Vesicles (blistering) BS Burning or stinging (sensation as
described by the subject) I Itching (sensation as described by the
subject)
[0106] The overall scoring system involved an erythema rating scale
plus the addition of clinical descriptors adapted from Dykes P J,
et al. An evaluation of the irritancy potential of povidine iodine
solutions: comparison of subjective and objective assessment
techniques. Clin Exp Dermatol 1992; 17(4):246-9, which is hereby
incorporated by reference in its entirety. The severity of erythema
was rated on a scale of 0-5, along with the presence of other
clinical signs or symptoms. These descriptors were noted by letters
are then added to the numerical score (e.g., strong erythema
spreading outside the treatment site with subject reporting
scaling=3S).
[0107] Cranial Nerves I-VII: Evaluation of cranial nerves I-VII was
performed at treatment (pre-application), day 2, and weeks 2, 4,
and 8 (or early discontinuation). Scores for each cranial nerve was
captured as outlined in Table 10 (for examination procedures and
criteria, see Bates B. A guide to physical examination and history
taking J B Lippincott & Co. 6th edition (1995) Chapter
7:168-169, 172-175; Chapter 18:505-8, which is hereby incorporated
by reference in its entirety).
TABLE-US-00013 TABLE 10 Cranial Nerve Assessment Rating Description
1 Normal 2 Abnormal, not clinically significant 3 Abnormal,
clinically significant 4 Not assessed
[0108] Facial Nerve Grading System: The Regional House-Brackmann
Facial Nerve Grading System (see, Yen, T L, et al. Significance of
House-Brackmann facial nerve grading global score in the setting of
differential facial nerve function. 0 to 1 Neurotol 2003;
24(1):118-222, which is hereby incorporated by reference in its
entirety) was designed to evaluate synkinesis and the four major
branches of the facial nerve (VII) that innervates target and
adjacent musculature. Functionality of the facial nerve (VII) was
evaluated pre-application, day 2, and weeks 2, 4, and 8 (or early
discontinuation). Examination procedures and criteria are set forth
in Table 11.
TABLE-US-00014 TABLE 11 Regional House-Brackmann Facial Nerve
Grading System Forehead 1 Normal forehead movement 2 Slight
weakness in forehead movement 3 Obvious but not disfiguring
asymmetry with motion, symmetric at rest 4 Obvious weakness of
disfiguring asymmetry with motion, symmetric at rest 5 Barely
perceptible motion in forehead, asymmetric at rest 6 No movement
Eye 1 Normal eye closure 2 Mild weakness in eye closure 3 Obvious
weakness but able to close eyes 4 Unable to close eye with maximal
effort 5 Barely perceptible eyelid movement 6 No movement Midface 1
Normal midface movement 2 Slight weakness in midface movement 3
Obvious but not disfiguring weakness, symmetric at rest 4 Obvious
weakness and disfiguring asymmetry with motion, symmetric at rest 5
Barely perceptible motion in midface, asymmetric at rest 6 No
movement Mouth 1 Normal corner of mouth movement 2 Slight weakness
of corner of mouth movement 3 Obvious but not disfiguring weakness,
symmetric at rest 4 Obvious weakness and disfiguring asymmetry with
motion, symmetric at rest 5 Barely perceptible corner of mouth
movement, asymmetric at rest 6 No movement Synkinesis 1 None 2 Mild
- obvious but not disfiguring 3 Severe - disfiguring or interferes
with function
[0109] Adverse Events:
[0110] AEs were graded based on the CTEP-CTCAE version 4.03 for
severity, where applicable. Otherwise AEs were graded as mild,
moderate, severe, or life-threatening. AEs were evaluated
post-application at treatment (day 0), and on follow-up visits at
day 2 and weeks 2, 4, and 8 (or early discontinuation).
Safety Evaluations
[0111] Safety evaluations included the following tests: [0112]
clinical laboratory tests (hematology, chemistry, urinalysis) at
screening and week 8; [0113] local irritation of treatment areas at
screening, treatment (day 0, pre- and post-application), day 2, and
weeks 2, 4, and 8; [0114] cranial nerves I-VII assessment including
Regional House-Brackmann Facial Nerve Evaluation for cranial nerve
VII at treatment (day 0, pre-application), day 2, and weeks 2, 4,
and 8; [0115] adverse events (AEs) from treatment (day 0) through
final evaluation; [0116] concomitant therapy/medication from
screening through final evaluation; and [0117] urine pregnancy test
for women of childbearing potential at screening, treatment (day 0)
and final evaluation.
[0118] All week 8 assessments were conducted at early
discontinuation visit, as applicable.
Statistical Analysis
[0119] All statistical programming and analyses were performed
using SAS, versin 9.1 or higher.
[0120] Populations:
[0121] All randomized subjects were included in the summaries of
demographic and other baseline characteristics. Efficacy analyses
were carried out in the intent to treat (ITT) population, defined
as all randomized subjects who received either the test article or
placebo. When possible, the last observation carried forward (LOCF)
approach was used to impute missing efficacy data. Per Protocol
(PP) analyses were also performed. No imputations were utilized for
the PP analyses. The safety population included all randomized
subjects, exposed to either the test article or placebo, that
provided any post-treatment safety information.
[0122] Efficacy Analyses:
[0123] The primary efficacy endpoint was based on change from
baseline in total nasal symptom score at week 4 for treatment
versus control. Demonstration of efficacy required trending at
p<0.20, rather than direct statistical significance at other
thresholds given the sample size and study design. Comparisons were
made using a 2 tailed t-test. Two interim analyses of efficacy were
performed, the first when 4 week data became available for at least
30 subjects; and the second when 75% or more of subjects reached
week 4 (or early study discontinuation).
[0124] Secondary endpoints included the following with statistical
analyses, as with the primary endpoint, or by Inferential
statistics based on the Fisher's exact test CMH, or Pearson Chi
Square test. [0125] change from baseline to week 2, 4, and 8,
respectively, in individual nasal symptom scores for rhinorrhea,
nasal congestion, nasal itching and sneezing, recorded daily by
subject and reported at visits (pre- and post-allergen challenge
where applicable); [0126] change from baseline to week 2, 4, and 8,
respectively in RQLQ; and [0127] PNIF and PEF at week 2, 4, and 8
respectively (pre- and post-allergen challenge where
applicable).
[0128] Change from pre-treatment values also was evaluated. The
difference between PNIF and PEF was calculated and examined by
treatment as well ("PEF-PNIF differences").
[0129] Exploratory analyses using daily and mean weekly
subject-recorded values also were conducted. Additional exploratory
analyses evaluated composite endpoints based upon categories and
definitions specified as primary or secondary endpoints above.
These composites combined two endpoints, with response required on
both simultaneously.
[0130] Safety Analyses: All treatment-emergent AEs occurring during
the study were recorded and classified on the basis of MedDRA
terminology for the safety population. Treatment-emergent AEs were
summarized by treatment group, the number of subjects reporting
treatment-emergent AEs, system organ class, preferred term,
severity, relationship, and seriousness. Comparisons between
treatment groups were made by tabulating the frequency of subjects
with one or more treatment-emergent AEs during the study. The
Fisher's exact test was used to compare the proportion of subjects
in each treatment group who report any treatment-emergent AEs at a
significance level of 0.05.
[0131] Serious adverse events (SAEs) also were listed by subject,
and summarized by treatment group, severity, and relationship to
study treatment.
[0132] Additional safety assessments, including clinical laboratory
values and urine pregnancy tests, were summarized for all treated
subjects by treatment group. Outcomes of the local irritation of
treated areas assessments were tabulated by visit, along with the
outcomes of the cranial nerves I-VII assessments. Concomitant
therapies/medications used at screening and each study visit were
also summarized.
[0133] Sample Size Justification: Approximately 70 subjects were
enrolled and randomized 1:1 to either test article or placebo.
Based upon an estimated 12% difference between treatment and
placebo groups, based on historical data for injectable botulinum
toxin type A, approximately 35 subjects per group were required to
demonstrate trending toward efficacy (primary statistical
analyses).
Results
[0134] Interim results when 75% or more of subjects reached week 4
are set forth below in Tables 12 and 13. Table 12 represents
secondary efficacy analysis results, based on PEF-PNIF differences
at week 4, in the ITT population. In this population, the
within-group median was substituted for missing observations.
TABLE-US-00015 TABLE 12 Treatment Group Test Article Placebo Total
(25 ng/ml) Group Subjects Number of ITT Subjects 16 14 30 Subjects
Assessed by PNIF 16 14 30 and PEF t Week 4 Mean (SD) pre-challenge
337.0 433.2 381.9 PEF-PNIF difference (96.31) (119.70) (116.64) at
Week 4 p-value.sup.1 0.0213 Mean (SD) post-challenge 394.8 444.9
418.2 PEF-PNIF difference (130.71) (142.01) (136.11) at Week 4
p-value.sup.1 0.3222 .sup.1p-value compares the treatmetn group to
the placebo group using a two-sided t-test.
[0135] Table 13 represents secondary efficacy analysis results,
based on post-challenge total nasal symptom (TNS) score at baseline
(screening) and week 4, in the ITT population. In this population,
the within-group median was substituted for missing
observations.
TABLE-US-00016 TABLE 13 Treatment Group Formulation 24 Placebo
Total (25 ng/ml) Group Subjects Number of ITT Subjects 16 14 30
Subjects Assessed by Post-Challenge TNS at 16 14 30 Baseline Mean
(SD) post-challenge TNS at baseline 8.4 (2.42) 8.6 (2.71) 8.5
(2.52) p-value.sup.1 0.8873 Subjects Assessed by Post-Challenge TNS
at 16 14 30 Week 4 Mean (SD) post-challenge TNS at Week 4 4.4
(1.90) 5.9 (3.70) 5.1 (2.92) p-value.sup.2 0.2109 Subjects Assessed
by Post-Challenge TNS 16 14 30 at Baseline and Week 4 Mean (SD)
absolute change from baseline 4.4 (1.90) 5.9 (3.70) 5.1 (2.92) in
Post-Challenge TNS at Week 4 p-value.sup.1 0.2167 Mean (SD) percent
change from baseline in -3.8 (2.62) -2.5 (2.79) -3.2 (2.73)
Post-Challenge TNS at Week 4 p-value.sup.1 0.3630 .sup.1p-value
compares the treatment group to the placebo group using a two-sided
t-test.
[0136] As shown in the Tables, total nasal symptom scores varied
depending on subject and pollen count, whereas results after forced
allergen challenge were more consistent. The results displayed a
pattern consistent with the literature, in terms of the magnitude
of an effective signal.
* * * * *