U.S. patent application number 15/494819 was filed with the patent office on 2017-08-10 for botulinum toxin compositions.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Terrence J. Hunt.
Application Number | 20170224786 15/494819 |
Document ID | / |
Family ID | 37402617 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170224786 |
Kind Code |
A1 |
Hunt; Terrence J. |
August 10, 2017 |
BOTULINUM TOXIN COMPOSITIONS
Abstract
A high potency botulinum toxin pharmaceutical composition
comprising two excipients (such as albumin and sodium chloride) in
a weight to weight ratio of between about 1 and about 100.
Inventors: |
Hunt; Terrence J.;
(Temecula, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
37402617 |
Appl. No.: |
15/494819 |
Filed: |
April 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14055749 |
Oct 16, 2013 |
9629904 |
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15494819 |
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13566804 |
Aug 3, 2012 |
8580250 |
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14055749 |
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11195268 |
Aug 1, 2005 |
8323666 |
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13566804 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/10 20180101; A61K
47/02 20130101; A61K 47/42 20130101; A61P 25/06 20180101; A61P 1/16
20180101; C12Y 304/24069 20130101; A61P 21/02 20180101; A61P 25/00
20180101; A61P 21/00 20180101; A61P 25/02 20180101; A61K 38/4893
20130101; A61K 9/1658 20130101; A61K 9/1611 20130101; A61K 9/19
20130101; A61P 17/00 20180101; A61P 1/00 20180101; A61K 9/0019
20130101; A61P 27/02 20180101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 9/19 20060101 A61K009/19; A61K 9/16 20060101
A61K009/16 |
Claims
1. A pharmaceutical composition comprising: (a) a botulinum toxin;
(b) a first excipient, wherein the first excipient is an albumin,
and; (c) a second excipient, wherein the second excipient is sodium
chloride; (d) wherein the weight to weight ratio of the first
excipient to the second excipient present in the pharmaceutical
composition is between 0.7 and 5.6, or between 7.4 and 55.6.
2. The pharmaceutical composition of claim 1, wherein the botulinum
toxin is present as a botulinum toxin complex.
3. The pharmaceutical composition of claim 1, wherein the botulinum
toxin is present as a pure botulinum toxin.
4. The pharmaceutical composition of claim 1, wherein the first
excipient is a serum albumin.
5. The pharmaceutical composition of claim 1, wherein the first
excipient is recombinant albumin.
6. A process for making a pharmaceutical composition, the process
comprising the step of: (a) combining about 2.5 ng of a botulinum
toxin type A complex with an albumin and sodium chloride in a
weight to weight ration of the albumin to the sodium chloride in
the pharmaceutical composition of between 0.7 and 5.6, or between
7.4 and 55.6, to form a mixture, and; (b) vacuum drying the
mixture, to thereby obtain a pharmaceutical composition with a
potency after reconstitution of between about 70 units and about
130 units.
7. The process of claim 6, further comprising the step, before the
vacuum drying step, lyophilizing the mixture.
8. The pharmaceutical composition obtainable by the process of
claim 6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/055,749, which was filed on Oct. 16, 2013, now U.S. Pat. No.
9,629,904, which is a continuation of U.S. application Ser. No.
13/566,804 filed Aug. 3, 2012, now U.S. Pat. No. 8,580,250, which
is a continuation of U.S. application Ser. No. 11/195,268 filed
Aug. 1, 2005, now U.S. Pat. No. 8,323,666, the entire contents of
which is incorporated herein by reference.
BACKGROUND
[0002] The present invention relates to improved botulinum toxin
pharmaceutical compositions. In particular, the present invention
relates to botulinum toxin pharmaceutical compositions with an
increased potency.
[0003] A pharmaceutical composition is a formulation which contains
at least one active ingredient (such as for example a Clostridial
toxin, such as a botulinum neurotoxin) as well as, for example, one
or more excipients, buffers, carriers, stabilizers, preservatives
and/or bulking agents, and is suitable for administration to a
patient to achieve a desired effect or result. The pharmaceutical
compositions disclosed herein can have diagnostic, therapeutic,
cosmetic and/or research utility in various species, such as for
example in human patients or subjects.
[0004] For storage stability and convenience of handling, a
pharmaceutical composition can be formulated as a lyophilized (i.e.
freeze dried) or vacuum dried powder which can be reconstituted
with a suitable fluid, such as saline or water, prior to
administration to a patient. Alternately, the pharmaceutical
composition can be formulated as an aqueous solution or suspension.
A pharmaceutical composition can contain a proteinaceous active
ingredient. Unfortunately, a protein active ingredient can be very
difficult to stabilize (i.e. maintained in a state where loss of
biological activity is minimized), resulting therefore in a loss of
protein and/or loss of protein activity during the formulation,
reconstitution (if required) and during the period of storage prior
to use of a protein containing pharmaceutical composition. Protein
active ingredient stability problems can occur because of
denaturation, degradation, dimerization, and/or polymerization of
the protein. Various excipients, such as albumins, gelatins,
polysaccharides and amino acids (native or recombinant) have been
used with differing degrees of success to try and stabilize a
protein active ingredient present in a pharmaceutical composition.
Additionally, cryoprotectants such as alcohols have been used to
reduce protein denaturation under the freezing conditions of
lyophilization.
[0005] Albumins are small, abundant plasma proteins. Human serum
albumin has a molecular weight of about 69 kiloDaltons (kD) and has
been used as a non-active ingredient in a pharmaceutical
composition where it can serve as a bulk carrier and stabilizer of
certain protein active ingredients present in a pharmaceutical
composition.
[0006] The stabilization function of albumin in a pharmaceutical
composition can be present both during the multi-step formulation
of the pharmaceutical composition and upon the later reconstitution
of the formulated pharmaceutical composition. Thus, stability can
be imparted by albumin to a proteinaceous active ingredient in a
pharmaceutical composition by, for example, (1) reducing adhesion
(commonly referred to as "stickiness") of the protein active
ingredient to surfaces, such as the surfaces of laboratory
glassware, vessels, to the vial in which the pharmaceutical
composition is reconstituted and to the inside surface of a syringe
used to inject the pharmaceutical composition. Adhesion of a
protein active ingredient to surfaces can lead to loss of active
ingredient and to denaturation of the remaining retained protein
active ingredient, both of which reduce the total activity of the
active ingredient present in the pharmaceutical composition, and;
(2) reducing denaturation of the active ingredient which can occur
upon preparation of a low dilution solution of the active
ingredient.
[0007] As well as being able to stabilize a protein active
ingredient in a pharmaceutical composition, human serum albumin
also has the advantage of generally negligible immunogenicity when
injected into a human patient. A compound with an appreciable
immunogenicity can cause the production of antibodies against it
which can lead to an anaphylactic reaction and/or to the
development of drug resistance, with the disease or disorder to be
treated thereby becoming potentially refractory to the
pharmaceutical composition which has an immunogenic component.
Gelatin has been used in some protein active ingredient
pharmaceutical compositions as an albumin substitute.
[0008] Botulinum Toxin
[0009] The anaerobic, gram positive bacterium Clostridium botulinum
produces a potent polypeptide neurotoxin, botulinum toxin, which
causes a neuroparalytic illness in humans and animals referred to
as botulism. Clostridium botulinum and its spores are commonly
found in soil and the bacterium can grow in improperly sterilized
and sealed food containers of home based canneries, which are the
cause of many of the cases of botulism. The effects of botulism
typically appear 18 to 36 hours after eating the foodstuffs
infected with a Clostridium botulinum culture or spores. The
botulinum toxin can apparently pass unattenuated through the lining
of the gut and attack peripheral motor neurons. Symptoms of
botulinum toxin intoxication can progress from difficulty walking,
swallowing, and speaking to paralysis of the respiratory muscles
and death.
[0010] Botulinum toxin type A is the most lethal natural biological
agent known to man. About 50 picograms of botulinum toxin (purified
neurotoxin complex) type A is a LD.sub.50 in mice. Interestingly,
on a molar basis, botulinum toxin type A is 1.8 billion times more
lethal than diphtheria, 600 million times more lethal than sodium
cyanide, 30 million times more lethal than cobrotoxin and 12
million times more lethal than cholera. Singh, Critical Aspects of
Bacterial Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins
II, edited by B. R. Singh et al., Plenum Press, New York (1976)
(where the stated LD.sub.50 of botulinum toxin type A of 0.3 ng
equals 1 U is corrected for the fact that about 0.05 ng of
BOTOX.RTM. equals 1 unit). One unit (U) of botulinum toxin is
defined as the LD.sub.50 upon intraperitoneal injection into female
Swiss Webster mice weighing 18-20 grams each. In other words, one
unit of botulinum toxin is the amount of botulinum toxin that kills
50% of a group of female Swiss Webster mice. Seven generally
immunologically distinct botulinum neurotoxins have been
characterized, these being respectively botulinum neurotoxin
serotypes A, B, C.sub.1, D, E, F, and G, each of which is
distinguished by neutralization with type-specific antibodies. The
different serotypes of botulinum toxin vary in the animal species
that they affect and in the severity and duration of the paralysis
they evoke. For example, it has been determined that botulinum
toxin type A is 500 times more potent, as measured by the rate of
paralysis produced in the rat, than is botulinum toxin type B.
Additionally, botulinum toxin type B has been determined to be
non-toxic in primates at a dose of 480 U/kg which is about 12 times
the primate LD.sub.50 for botulinum toxin type A. The botulinum
toxins apparently bind with high affinity to cholinergic motor
neurons, are translocated into the neuron and block the presynaptic
release of acetylcholine.
[0011] Botulinum toxins have been used in clinical settings for the
treatment of neuromuscular disorders characterized by hyperactive
skeletal muscles. Botulinum toxin type A was approved by the U.S.
Food and Drug Administration in 1989 for the treatment of essential
blepharospasm, strabismus and hemifacial spasm in patients over the
age of twelve. Clinical effects of peripheral injection (i.e.
intramuscular or subcutaneous) botulinum toxin type A are usually
seen within one week of injection, and often within a few hours
after injection. The typical duration of symptomatic relief (i.e.
flaccid muscle paralysis) from a single intramuscular injection of
botulinum toxin type A can be about three months to about six
months.
[0012] Although all the botulinum toxins serotypes apparently
inhibit release of the neurotransmitter acetylcholine at the
neuromuscular junction, they do so by affecting different
neurosecretory proteins and/or cleaving these proteins at different
sites. Botulinum toxin A is a zinc endopeptidase which can
specifically hydrolyze a peptide linkage of the intracellular,
vesicle associated protein SNAP-25. Botulinum type E also cleaves
the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25),
but targets different amino acid sequences within this protein, as
compared to botulinum toxin type A. Botulinum toxin types B, D, F
and G act on vesicle-associated protein (VAMP, also called
synaptobrevin), with each serotype cleaving the protein at a
different site. Finally, botulinum toxin type C.sub.1 has been
shown to cleave both syntaxin and SNAP-25. These differences in
mechanism of action may affect the relative potency and/or duration
of action of the various botulinum toxin serotypes.
[0013] Regardless of serotype, the molecular mechanism of toxin
intoxication appears to be similar and to involve at least three
steps or stages. In the first step of the process, the toxin binds
to the presynaptic membrane of the target neuron through a specific
interaction between the heavy chain (H chain) and a cell surface
receptor; the receptor is thought to be different for each serotype
of botulinum toxin and for tetanus toxin. The carboxyl end segment
of the H chain, H.sub.C, appears to be important for targeting of
the toxin to the cell surface.
[0014] In the second step, the toxin crosses the plasma membrane of
the poisoned cell. The toxin is first engulfed by the cell through
receptor-mediated endocytosis, and an endosome containing the toxin
is formed. The toxin then escapes the endosome into the cytoplasm
of the cell. This last step is thought to be mediated by the amino
end segment of the H chain, H.sub.N, which triggers a
conformational change of the toxin in response to a pH of about 5.5
or lower. Endosomes are known to possess a proton pump which
decreases intra endosomal pH. The conformational shift exposes
hydrophobic residues in the toxin, which permits the toxin to embed
itself in the endosomal membrane. The toxin then translocates
through the endosomal membrane into the cytosol.
[0015] The last step of the mechanism of botulinum toxin activity
appears to involve reduction of the disulfide bond joining the H
and L chain. The entire toxic activity of botulinum and tetanus
toxins is contained in the L chain of the holotoxin; the L chain is
a zinc (Zn++) endopeptidase which selectively cleaves proteins
essential for recognition and docking of
neurotransmitter-containing vesicles with the cytoplasmic surface
of the plasma membrane, and fusion of the vesicles with the plasma
membrane. Tetanus neurotoxin, botulinum toxin B, D, F, and G cause
degradation of synaptobrevin (also called vesicle-associated
membrane protein (VAMP)), a synaptosomal membrane protein. Most of
the VAMP present at the cytosolic surface of the synaptic vesicle
is removed as a result of any one of these cleavage events. Each
toxin specifically cleaves a different bond.
[0016] The molecular weight of the botulinum toxin protein
molecule, for all seven of the known botulinum toxin serotypes, is
about 150 kD. Interestingly, the botulinum toxins are released by
Clostridial bacterium as complexes comprising the 150 kD botulinum
toxin protein molecule along with associated non-toxin proteins.
Thus, the botulinum toxin type A complex can be produced by
Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum
toxin types B and C.sub.1 are apparently produced as only a 500 kD
complex. Botulinum toxin type D is produced as both 300 kD and 500
kD complexes. Finally, botulinum toxin types E and F are produced
as only approximately 300 kD complexes. The complexes (i.e.
molecular weight greater than about 150 kD) are believed to contain
a non-toxin hemagglutinin protein and a non-toxin and non-toxic
nonhemagglutinin protein. These two non-toxin proteins (which along
with the botulinum toxin molecule can comprise the relevant
neurotoxin complex) may act to provide stability against
denaturation to the botulinum toxin molecule and protection against
digestive acids when toxin is ingested. Additionally, it is
possible that the larger (greater than about 150 kD molecular
weight) botulinum toxin complexes may result in a slower rate of
diffusion of the botulinum toxin away from a site of intramuscular
injection of a botulinum toxin complex. The toxin complexes can be
dissociated into toxin protein and hemagglutinin proteins by
treating the complex with red blood cells at pH 7.3. The toxin
protein has a marked instability upon removal of the hemagglutinin
protein.
[0017] All the botulinum toxin serotypes are made by Clostridium
botulinum bacteria as inactive single chain proteins which must be
cleaved or nicked by proteases to become neuroactive. The bacterial
strains that make botulinum toxin serotypes A and G possess
endogenous proteases and serotypes A and G can therefore be
recovered from bacterial cultures in predominantly their active
form. In contrast, botulinum toxin serotypes C.sub.1, D, and E are
synthesized by nonproteolytic strains and are therefore typically
unactivated when recovered from culture. Serotypes B and F are
produced by both proteolytic and nonproteolytic strains and
therefore can be recovered in either the active or inactive form.
However, even the proteolytic strains that produce, for example,
the botulinum toxin type B serotype only cleave a portion of the
toxin produced. The exact proportion of nicked to unnicked
molecules depends on the length of incubation and the temperature
of the culture. Therefore, a certain percentage of any preparation
of, for example, the botulinum toxin type B toxin is likely to be
inactive, possibly accounting for the known significantly lower
potency of botulinum toxin type B as compared to botulinum toxin
type A. The presence of inactive botulinum toxin molecules in a
clinical preparation will contribute to the overall protein load of
the preparation, which has been linked to increased antigenicity,
without contributing to its clinical efficacy. Additionally, it is
known that botulinum toxin type B has, upon intramuscular
injection, a shorter duration of activity and is also less potent
than botulinum toxin type A at the same dose level.
[0018] In vitro studies have indicated that botulinum toxin
inhibits potassium cation induced release of both acetylcholine and
norepinephrine from primary cell cultures of brainstem tissue.
Additionally, it has been reported that botulinum toxin inhibits
the evoked release of both glycine and glutamate in primary
cultures of spinal cord neurons and that in brain synaptosome
preparations botulinum toxin inhibits the release of each of the
neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and
glutamate.
[0019] High quality crystalline botulinum toxin type A can be
produced from the Hall A strain of Clostridium botulinum with
characteristics of .gtoreq.3.times.10.sup.7 U/mg, an
A.sub.260/A.sub.278 of less than 0.60 and a distinct pattern of
banding on gel electrophoresis. The known Schantz process can be
used to obtain crystalline botulinum toxin type A, as set forth in
Schantz, E. J., et al, Properties and use of Botulinum toxin and
Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56: 80-99
(1992). Generally, the botulinum toxin type A complex can be
isolated and purified from an anaerobic fermentation by cultivating
Clostridium botulinum type A in a suitable medium. Raw toxin can be
harvested by precipitation with sulfuric acid and concentrated by
ultramicrofiltration. Purification can be carried out by dissolving
the acid precipitate in calcium chloride. The toxin can then be
precipitated with cold ethanol. The precipitate can be dissolved in
sodium phosphate buffer and centrifuged. Upon drying there can then
be obtained approximately 900 kD crystalline botulinum toxin type A
complex with a specific potency of 3.times.10.sup.7 LD.sub.50 U/mg
or greater. This known process can also be used, upon separation
out of the non-toxin proteins, to obtain pure botulinum toxins,
such as for example: purified botulinum toxin type A with an
approximately 150 kD molecular weight with a specific potency of
1-2.times.10.sup.8 LD.sub.50 U/mg or greater; purified botulinum
toxin type B with an approximately 156 kD molecular weight with a
specific potency of 1-2.times.10.sup.8 LD.sub.50 U/mg or greater,
and; purified botulinum toxin type F with an approximately 155 kD
molecular weight with a specific potency of 1-2.times.10.sup.7
LD.sub.50 U/mg or greater.
[0020] A pure (i.e. 150 kDa botulinum toxin free of the non-toxin
complex proteins) can be obtained by loading a solution of a
botulinum toxin complex onto a suitable ion exchange chromatograph
in a pH 8 buffer to disassociate the non toxin complex proteins
from the 150 kDa botulinum toxin molecule, thereby providing (in
the flow through from the column) a solution of a botulinum toxin
neurotoxic component with an approximately 150 kD molecular
weight.
[0021] Pure botulinum toxin (i.e. the approximately 150 kDa
molecular weight neurotoxic component of a botulinum toxin complex)
has been used to treat humans. See e.g. Kohl A., et al., Comparison
of the effect of botulinum toxin A (Botox.RTM.) with the
highly-purified neurotoxin (NT 201) in the extensor digitorum
brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, a
botulinum toxin pharmaceutical composition can be prepared using a
pure (approx 150 kDa) botulinum toxin, as opposed to use of a
botulinum toxin complex. A pure botulinum toxin type A is available
from Merz Pharmaceuticals under the tradename XEOMIN.
[0022] Already prepared and purified botulinum toxins and toxin
complexes suitable for preparing pharmaceutical formulations can be
obtained from List Biological Laboratories, Inc., Campbell, Calif.;
the Centre for Applied Microbiology and Research, Porton Down,
U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of St
Louis, Mo.
[0023] Examples of clinical use of a botulinum toxin are:
(1) about 75-125 units of BOTOX.RTM..sup.1 per intramuscular
injection (multiple muscles) to treat cervical dystonia;
.sup.1Available from Allergan, Inc., of Irvine, Calif. under the
tradename BOTOX.RTM.. (2) 5-10 units of BOTOX.RTM. per
intramuscular injection to treat glabellar lines (brow furrows) (5
units injected intramuscularly into the procerus muscle and 10
units injected intramuscularly into each corrugator supercilii
muscle); (3) about 30-80 units of BOTOX.RTM. to treat constipation
by intrasphincter injection of the puborectalis muscle; (4) about
1-5 units per muscle of intramuscularly injected BOTOX.RTM. to
treat blepharospasm by injecting the lateral pre-tarsal orbicularis
oculi muscle of the upper lid and the lateral pre-tarsal
orbicularis oculi of the lower lid. (5) to treat strabismus,
extraocular muscles have been injected intramuscularly with between
about 1-5 units of BOTOX.RTM., the amount injected varying based
upon both the size of the muscle to be injected and the extent of
muscle paralysis desired (i.e. amount of diopter correction
desired). (6) to treat upper limb spasticity following stroke by
intramuscular injections of BOTOX.RTM. into five different upper
limb flexor muscles, as follows:
[0024] (a) flexor digitorum profundus: 7.5 U to 30 U
[0025] (b) flexor digitorum sublimis: 7.5 U to 30 U
[0026] (c) flexor carpi ulnaris: 10 U to 40 U
[0027] (d) flexor carpi radialis: 15 U to 60 U
[0028] (e) biceps brachii: 50 U to 200 U. Each of the five
indicated muscles has been injected at the same treatment session,
so that the patient receives from 90 U to 360 U of upper limb
flexor muscle BOTOX.RTM. by intramuscular injection at each
treatment session.
(7) to treat migraine, pericranial injected (injected symmetrically
into glabellar, frontalis and temporalis muscles) injection of 25 U
of BOTOX.RTM. has showed significant benefit as a prophylactic
treatment of migraine compared to vehicle as measured by decreased
measures of migraine frequency, maximal severity, associated
vomiting and acute medication use over the three month period
following the 25 U injection.
[0029] It is known that botulinum toxin type A can have an efficacy
for up to 12 months (European J. Neurology 6 (Supp 4):
S111-S1150:1999), and in some circumstances for as long as 27
months. The Laryngoscope 109:1344-1346:1999. However, the usual
duration of the effect of an intramuscular injection of Botox.RTM.
is typically about 3 to 4 months.
[0030] The success of botulinum toxin type A to treat a variety of
clinical conditions has led to interest in other botulinum toxin
serotypes. European patent EP1112082 ("Stable liquid formulations
of botulinum toxin"), issued Jul. 31, 2002 claims a stable liquid
pharmaceutical botulinum toxin formulation comprising a buffer (pH
5-6) and a botulinum toxin, wherein the toxin formulation is stable
as a liquid for at least one year at temperatures between 0-10 C or
at least 6 months at temperatures between 10 and 30 C. Such a
botulinum toxin pharmaceutical formulation (an embodiment of which
is sold commercially under the tradename MyoBloc.RTM. or
NeuroBloc.RTM. by Solstice Neurosciences, Inc., of San Diego,
Calif.) is prepared as a liquid solution (no lyophilization or
vacuum drying is carried out) which does not require reconstitution
before use.
[0031] Chinese patent application CN 1215084A discusses an albumin
free botulinum toxin type A formulated with gelatin, an animal
derived protein. U.S. Pat. No. 6,087,327 also discloses a
composition of botulinum toxin types A and B formulated with
gelatin.
[0032] U.S. Pat. No. 5,512,547 (Johnson et al) entitled
"Pharmaceutical Composition of Botulinum Neurotoxin and Method of
Preparation" issued Apr. 30, 1996 and claims a pure botulinum type
A formulation comprising albumin and trehalose, storage stable at
37 degrees C.
[0033] U.S. Pat. No. 5,756,468 (Johnson et al) issued May 26, 1998
("Pharmaceutical Compositions of Botulinum Toxin or Botulinum
Neurotoxin and Method of Preparation"), and claims a lyophilized
botulinum toxin formulation comprising a thioalkyl, albumin and
trehalose which can be stored between 25 degrees C. and 42 degrees
C.
[0034] U.S. Pat. No. 5,696,077 (Johnson et al) entitled
"Pharmaceutical Composition Containing Botulinum B Complex" issued
Dec. 9, 1997 and claims a freeze dried, sodium chloride-free
botulinum type B complex formation comprising a type B complex and
a protein excipient.
[0035] U.S. patent application publication number 2003 0118598
(Hunt) discloses uses of various excipients such as a recombinant
albumin, collagen or a starch to stabilize a botulinum toxin.
[0036] Goodnough M. C., et al., Stabilization of botulinum toxin
type A during lyophilization, Appl Environ Microbiol 1992;
58(10):3426-3428, and; Goodnough M. C., et al., Recovery of type-A
botulinal toxin following lyophilization, Acs Symposium Series
1994; 567(-):193-203, disclose botulinum toxin formulations
comprising albumin and sodium chloride in a ratio of about 0.6:1
(i.e. 5 mg of BSA or HSA per ml and 9 mg of NaCl per ml of
reconstituted botulinum toxin solution) and state that elimination
of sodium chloride from the botulinum toxin formulation (and
increasing the HSA to as much as 9 mg/ml in the salt free
formulations) contributed significantly to obtaining a formulation
with active botulinum toxin.
[0037] The botulinum toxin molecule (about 150 kDa), as well as the
botulinum toxin complexes (about 300-900 kDa), such as the toxin
type A complex are extremely susceptible to denaturation due to
surface denaturation, heat, and alkaline conditions. Inactivated
toxin forms toxoid proteins which may be immunogenic. The resulting
antibodies can render a patient refractory to toxin injection.
[0038] As with enzymes generally, the biological activities of the
botulinum toxins (which are intracellular peptidases) are
dependant, at least in part, upon their three dimensional
conformation. Thus, botulinum toxin type A is detoxified by heat,
various chemicals surface stretching and surface drying.
Additionally, it is known that dilution of the toxin complex
obtained by the known culturing, fermentation and purification to
the much, much lower toxin concentrations used for pharmaceutical
composition formulation results in rapid detoxification of the
toxin unless a suitable stabilizing agent is present. Dilution of
the toxin from milligram quantities to a solution containing
nanograms per milliliter presents significant difficulties because
of the rapid loss of specific toxicity upon such great dilution.
Since the botulinum toxin may be used months or years after the
botulinum toxin containing pharmaceutical composition is
formulated, the botulinum toxin must be stabilized with a
stabilizing agent, such as an albumin or gelatin. Additionally, for
storage stability botulinum toxin can be processed into a solid
state (i.e. a powder) by known lyophilization or vacuum-drying
techniques.
[0039] Furthermore, any one of the harsh pH, temperature and
concentration range conditions required to lyophilize (freeze-dry)
or vacuum dry a botulinum toxin containing pharmaceutical
composition into a toxin shipping and storage format (ready for use
or reconstitution by a physician) can detoxify the toxin. Thus,
gelatin and serum albumin have been used with some success to
stabilize botulinum toxin.
[0040] A commercially available botulinum toxin containing
pharmaceutical composition is sold under the trademark BOTOX.RTM.
(available from Allergan, Inc., of Irvine, Calif.). BOTOX.RTM.
consists of a purified botulinum toxin type A complex, human serum
albumin, and sodium chloride packaged in sterile, vacuum-dried
form. The botulinum toxin type A is made from a culture of the Hall
strain of Clostridium botulinum grown in a medium containing N--Z
amine and yeast extract. The botulinum toxin type A complex is
purified from the culture solution by a series of acid
precipitations to a crystalline complex consisting of the active
high molecular weight toxin protein and an associated hemagglutinin
protein. The crystalline complex is re-dissolved in a solution
containing saline and albumin and sterile filtered (0.2 microns)
prior to vacuum-drying. BOTOX.RTM. can be reconstituted with
sterile, non-preserved saline prior to intramuscular injection.
Each vial of BOTOX.RTM. contains about 100 units (U) of Clostridium
botulinum toxin type A complex, 0.5 milligrams of human serum
albumin and 0.9 milligrams of sodium chloride in a sterile,
vacuum-dried form without a preservative.
[0041] To reconstitute vacuum-dried BOTOX.RTM. sterile normal
saline without a preservative (0.9% Sodium Chloride injection) is
used by drawing up the proper amount of diluent in the appropriate
size syringe. Since BOTOX.RTM. is denatured by bubbling or similar
violent agitation, the diluent is gently injected into the vial.
For sterility reasons, BOTOX.RTM. should be administered within
about 72 hours after reconstitution. During this time period,
reconstituted BOTOX.RTM. is stored in a refrigerator (2.degree. to
8.degree. C.). Reconstituted BOTOX.RTM. is clear, colorless and
free of particulate matter. The vacuum-dried product is stored in a
freezer or refrigerator.
[0042] Other commercially available botulinum toxin containing
pharmaceutical compositions include Dysport.RTM. (Clostridium
botulinum type A toxin hemagglutinin complex with human serum
albumin and lactose in the formulation, available from Ipsen
Limited, Berkshire, U.K. as a powder to be reconstituted with 0.9%
sodium chloride before use), and MyoBloc.TM. (an injectable
solution comprising botulinum toxin type B, human serum albumin,
sodium succinate, and sodium chloride at about pH 5.6, available
from Elan Corporation, Dublin, Ireland).
[0043] It has been reported that a suitable alternative to human
serum albumin as a botulinum toxin stabilizer may be another
protein or alternatively a low molecular weight (non-protein)
compound. Carpender et al., Interactions of Stabilizing Additives
with Proteins During Freeze-Thawing and Freeze-Drying,
International Symposium on Biological Product Freeze-Drying and
Formulation, 24-26 Oct. 1990; Karger (1992), 225-239.
[0044] Human serum albumin is believed to function in a
pharmaceutical composition as more than a mere bulking agent. Thus,
albumin apparently can interact with botulinum toxin so as to
increase the potency of the neurotoxin. For example, it is known
that bovine serum albumin can act as more than a mere stabilizing
excipient for botulinum toxin type A, since bovine serum albumin
apparently also accelerates the rate of catalysis of synthetic
peptide substrates, which substrates resemble the SNAP-25
intraneuronal substrate for botulinum toxin type A Schmidt, et al.,
Endoproteinase Activity of Type A Botulinum Neurotoxin Substrate
Requirements and Activation by Serum Albumin, J. of Protein
Chemistry, 16 (1), 19-26 (1997). Thus, albumin may have a
potentiating effect, apparently by affecting rate kinetics, upon
the intracellular proteolytic action of a botulinum toxin upon the
toxin's substrate. This potentiating effect may be due to albumin
which has accompanied the botulinum toxin upon endocytosis of the
toxin into a target neuron or the potentiating effect may be due to
the pre-existing presence cytoplasmic albumin within the neuron
protein prior to endocytosis of the botulinum toxin.
[0045] Acetylcholine
[0046] Typically only a single type of small molecule
neurotransmitter is released by each type of neuron in the
mammalian nervous system. The neurotransmitter acetylcholine is
secreted by neurons in many areas of the brain, but specifically by
the large pyramidal cells of the motor cortex, by several different
neurons in the basal ganglia, by the motor neurons that innervate
the skeletal muscles, by the preganglionic neurons of the autonomic
nervous system (both sympathetic and parasympathetic), by the
postganglionic neurons of the parasympathetic nervous system, and
by some of the postganglionic neurons of the sympathetic nervous
system. Essentially, only the postganglionic sympathetic nerve
fibers to the sweat glands, the piloerector muscles and a few blood
vessels are cholinergic as most of the postganglionic neurons of
the sympathetic nervous system secret the neurotransmitter
norepinephrine. In most instances acetylcholine has an excitatory
effect. However, acetylcholine is known to have inhibitory effects
at some of the peripheral parasympathetic nerve endings, such as
inhibition of heart rate by the vagal nerve.
[0047] The efferent signals of the autonomic nervous system are
transmitted to the body through either the sympathetic nervous
system or the parasympathetic nervous system. The preganglionic
neurons of the sympathetic nervous system extend from preganglionic
sympathetic neuron cell bodies located in the intermediolateral
horn of the spinal cord. The preganglionic sympathetic nerve
fibers, extending from the cell body, synapse with postganglionic
neurons located in either a paravertebral sympathetic ganglion or
in a prevertebral ganglion. Since, the preganglionic neurons of
both the sympathetic and parasympathetic nervous system are
cholinergic, application of acetylcholine to the ganglia will
excite both sympathetic and parasympathetic postganglionic
neurons.
[0048] Acetylcholine activates two types of receptors, muscarinic
and nicotinic receptors. The muscarinic receptors are found in all
effector cells stimulated by the postganglionic, neurons of the
parasympathetic nervous system as well as in those stimulated by
the postganglionic cholinergic neurons of the sympathetic nervous
system. The nicotinic receptors are found in the adrenal medulla,
as well as within the autonomic ganglia, that is on the cell
surface of the postganglionic neuron at the synapse between the
preganglionic and postganglionic neurons of both the sympathetic
and parasympathetic systems. Nicotinic receptors are also found in
many nonautonomic nerve endings, for example in the membranes of
skeletal muscle fibers at the neuromuscular junction.
[0049] Acetylcholine is released from cholinergic neurons when
small, clear, intracellular vesicles fuse with the presynaptic
neuronal cell membrane. A wide variety of non-neuronal secretory
cells, such as, adrenal medulla (as well as the PC12 cell line) and
pancreatic islet cells release catecholamines and parathyroid
hormone, respectively, from large dense-core vesicles. The PC12
cell line is a clone of rat pheochromocytoma cells extensively used
as a tissue culture model for studies of sympathoadrenal
development. Botulinum toxin inhibits the release of both types of
compounds from both types of cells in vitro, permeabilized (as by
electroporation) or by direct injection of the toxin into the
denervated cell. Botulinum toxin is also known to block release of
the neurotransmitter glutamate from cortical synaptosomes cell
cultures.
[0050] A neuromuscular junction is formed in skeletal muscle by the
proximity of axons to muscle cells. A signal transmitted through
the nervous system results in an action potential at the terminal
axon, with activation of ion channels and resulting release of the
neurotransmitter acetylcholine from intraneuronal synaptic
vesicles, for example at the motor endplate of the neuromuscular
junction. The acetylcholine crosses the extracellular space to bind
with acetylcholine receptor proteins on the surface of the muscle
end plate. Once sufficient binding has occurred, an action
potential of the muscle cell causes specific membrane ion channel
changes, resulting in muscle cell contraction. The acetylcholine is
then released from the muscle cells and metabolized by
cholinesterases in the extracellular space. The metabolites are
recycled back into the terminal axon for reprocessing into further
acetylcholine.
[0051] Thus, what is needed is a process for preparing a botulinum
toxin pharmaceutical formulation where little or no botulinum toxin
is lost during the compounding process. Alternately stated, what is
needed is a process for preparing a botulinum toxin pharmaceutical
formulation which permits a high recovery of the botulinum toxin
after reconstitution. Loss of botulinum toxin during compounding
(which leads to a lower recovery) presents the possibility of
inactivated toxin (toxoid) being present in the final reconstituted
product, thereby raising an antigenic potential of the product upon
administration to a patient. The theoretical optimal is to have
100% of the botulinum toxin which enters the compounding process
present in the final reconstituted product and present as
biologically active botulinum toxin.
[0052] What is also needed therefore is a botulinum toxin
containing pharmaceutical composition with a higher potency, as
compared to the potencies of known botulinum toxin pharmaceutical
compositions. Expressed in an alternate manner, what is needed is a
botulinum toxin pharmaceutical composition with a higher potency of
the botulinum toxin for each nanogram of the botulinum toxin
present in the botulinum toxin pharmaceutical composition.
SUMMARY OF THE INVENTION
[0053] The present invention meets this need by providing a process
for preparing a botulinum toxin pharmaceutical formulation wherein
little or no botulinum toxin is lost during the compounding
process. Alternately stated, a process within the scope of the
present invention permits preparation of a botulinum toxin
pharmaceutical formulation with a high recovery of the botulinum
toxin after reconstitution. Significantly, processes within the
scope of the present invention approach the theoretical optimum by
permitting about 100% of the botulinum toxin which enters the
compounding process to be present in the final reconstituted
product as biologically active botulinum toxin.
[0054] Additionally the present invention meets the needs expressed
above by providing a botulinum toxin pharmaceutical composition
with a higher (that is an increased) potency, as compared to the
potencies of known botulinum toxin pharmaceutical compositions. In
particular, the present invention meets this need by providing a
powdered (due for example to freeze drying, lyophilization and/or
vacuum drying) botulinum toxin pharmaceutical composition which
upon (i.e. after) reconstitution with an aqueous fluid (such as
saline or water) has an increased potency (as determined for
example by the mouse LD.sub.50 assay), as compared to the potency
after reconstitution with an aqueous fluid of a known powdered
botulinum toxin pharmaceutical composition (such as Botox.RTM. or
Dysport.RTM.). Potency upon reconstitution can be referred to as
potency after "recovery". Hence, the present invention includes a
botulinum toxin pharmaceutical composition with an increased
potency upon recovery, or synonymously with an increased recovered
or recovery potency.
[0055] Briefly, an important aspect of the present invention for
meeting these dual needs of a high conservation (i.e. low or small
loss) of the amount of the botulinum toxin which enters a
compounding process (as compared to the amount of active botulinum
toxin present in the final [compounded] product), and a high
botulinum toxin pharmaceutical composition recovered potency, is
achieved by compounding the solid form botulinum toxin
pharmaceutical composition such that two of the excipients present
in the botulinum toxin pharmaceutical composition are present, at
least during the compounding process, in a particular weight to
weight ratio or in a particular weight to weight ratio range.
Definitions
[0056] As used herein, the words or terms set forth below have the
following definitions.
[0057] "About" means that the item, parameter or term so qualified
encompasses a range of plus or minus ten percent above and below
the value of the stated item, parameter or term.
[0058] "Administration", or "to administer" means the step of
giving (i.e. administering) a pharmaceutical composition to a
subject. The pharmaceutical compositions disclosed herein are
"locally administered". Systemic (i.e. intravenous or oral) routes
of administration are excluded from the scope of the present
invention, to the extent that a systemic administration would
result in systemic effects of a systemically administered active
ingredient. Systemic administration of a targeted active ingredient
which does not result in systemic effects is not excluded from the
scope of the present invention (see e.g. published U.S. patent
applications 20040086532 and 20040086531). Local administration
includes, but is not limited to, intramuscular (i.m.)
administration, intradermal or subdermal administration,
subcutaneous administration, intrathecal administration,
intraperitoneal (i.p.) administration, topical contact, and
implantation of a slow-release device such as polymeric implant or
miniosmotic pump.
[0059] "Botulinum toxin" means a neurotoxin produced by Clostridium
botulinum, as well as a botulinum toxin (or the light chain or the
heavy chain thereof) made recombinantly by a non-Clostridial
species. The phrase "botulinum toxin", as used herein, encompasses
the botulinum toxin serotypes A, B, C, D, E, F and G. Botulinum
toxin, as used herein, also encompasses both a botulinum toxin
complex (i.e. the 300, 600 and 900 kDa complexes) as well as the
purified botulinum toxin (i.e. about 150 kDa). "Purified (or pure)
botulinum toxin" is defined as a botulinum toxin that is isolated,
or substantially isolated, from other proteins, including proteins
that form a botulinum toxin complex. A purified (or pure) botulinum
toxin can be greater than 95% pure, and preferably is greater than
99% pure.
[0060] "Clostridial neurotoxin" means a neurotoxin produced from,
or native to, a Clostridial bacterium, such as Clostridium
botulinum, Clostridium butyricum or Clostridium baratii, as well as
a Clostridial neurotoxin made recombinantly by a non-Clostridial
species.
[0061] "Enhanced potency" with regard to a botulinum toxin
containing pharmaceutical composition means that the composition
has a potency (as determined, for example, by the mouse LD.sub.50
assay) which is from at least 5% and up to 50%, or more, greater
than the potency of a reference botulinum toxin pharmaceutical
composition. A reference botulinum toxin pharmaceutical composition
can contain a botulinum toxin, sodium chloride and HSA, wherein the
albumin and the sodium chloride are present in a weight to weight
ratio of about 0.6:1.
[0062] "Entirely free (i.e. "consisting of" terminology) means that
within the detection range of the instrument or process being used,
the substance cannot be detected or its presence cannot be
confirmed.
[0063] "Essentially free" (or "consisting essentially of") means
that only trace amounts of the substance can be detected.
[0064] "Excipient" means a substance present in a pharmaceutical
composition other than the active pharmaceutical ingredient present
in the pharmaceutical composition. An excipient can be a buffer,
carrier, stabilizer, preservative, diluent, vehicle, and/or a
bulking agent, such as an albumin, gelatin, collagen and/or sodium
chloride.
[0065] "Immobilizing" means a step that prevents a subject from
moving one or more body parts. If a sufficient number of body parts
are immobilized, the subject will accordingly be immobilized. Thus,
"immobilizing" encompasses the immobilization of a body part, such
as a limb, and/or the complete immobilization of a subject.
[0066] "Modified botulinum toxin" means a botulinum toxin that has
had at least one of its amino acids deleted, modified, or replaced,
as compared to a native botulinum toxin. Additionally, the modified
botulinum toxin can be a recombinantly produced neurotoxin, or a
derivative or fragment of a recombinantly made neurotoxin. A
modified botulinum toxin retains at least one biological activity
of the native botulinum toxin, such as, the ability to bind to a
botulinum toxin receptor, or the ability to inhibit
neurotransmitter release from a neuron. One example of a modified
botulinum toxin is a botulinum toxin that has a light chain from
one botulinum toxin serotype (such as serotype A), and a heavy
chain from a different botulinum toxin serotype (such as serotype
B). Another example of a modified botulinum toxin is a botulinum
toxin coupled to a neurotransmitter, such as substance P.
[0067] "Patient" means a human or non-human subject receiving
medical or veterinary care. Accordingly, as disclosed herein, the
compositions may be used in treating any animal, such as
mammals.
[0068] "Pharmaceutical composition" means a formulation in which an
active ingredient can be a neurotoxin, such as a Clostridial
neurotoxin. The word "formulation" means that there is at least one
additional ingredient in the pharmaceutical composition besides a
neurotoxin active ingredient. A pharmaceutical composition is
therefore a formulation which is suitable for diagnostic,
therapeutic or cosmetic use (i.e. by intramuscular or subcutaneous
injection or by insertion of a depot or implant) to a subject, such
as a human patient. The pharmaceutical composition can be: in a
lyophilized or vacuum dried condition; a solution formed after
reconstitution of the lyophilized or vacuum dried pharmaceutical
composition with saline or water, or; as a solution which does not
require reconstitution. The neurotoxin active ingredient can be one
of the botulinum toxin serotypes A, B, C.sub.1, D, E, F or G or a
tetanus toxin, all of which can be made natively by Clostridial
bacteria. As stated, a pharmaceutical composition can be liquid or
solid, for example vacuum-dried. The constituent ingredients of a
pharmaceutical composition can be included in a single composition
(that is all the constituent ingredients, except for any required
reconstitution fluid, are present at the time of initial
compounding of the pharmaceutical composition) or as a
two-component system, for example a vacuum-dried composition
reconstituted with a diluent such as saline which diluent contains
an ingredient (such as water) not present in the initial
compounding of the pharmaceutical composition.
[0069] "Polysaccharide" means a polymer of more than two saccharide
molecule monomers, which monomers can be identical or
different.
[0070] "Protein stabilizer" (or "primary stabilizer") is a chemical
agent that assists to preserve or maintain the biological structure
(i.e. the three dimensional conformation) and/or biological
activity of a protein (such as a Clostridial neurotoxin, such as a
botulinum toxin). Stabilizers can be proteins or polysaccharides.
Examples of protein stabilizers include hydroxyethyl starch
(hetastarch), serum albumin, gelatin, collagen, as well as a
recombinant albumin, gelatin or collagen. As disclosed herein, the
primary stabilizer can be a synthetic agent that would not produce
an immunogenic response (or produces an attenuated immune response)
in a subject receiving a composition containing the primary
stabilizer. In other embodiments of the invention, the protein
stabilizers may be proteins from the same species of animal that is
being administered the protein. Additional stabilizers may also be
included in a pharmaceutical composition. These additional or
secondary stabilizers may be used alone or in combination with
primary stabilizers, such as proteins and polysaccharides.
Exemplary secondary stabilizers include, but are not limited to
non-oxidizing amino acid derivatives (such as a tryptophan
derivative, such as N-acetyl-tryptophan ("NAT")), caprylate (i.e.
sodium caprylate), a polysorbate (i.e. P80), amino acids, and
divalent metal cations such as zinc. A pharmaceutical composition
can also include preservative agents such as benzyl alcohol,
benzoic acid, phenol, parabens and sorbic acid. A "recombinant
stabilizer" is a "primary stabilizer" made by recombinant means,
such as for example, a recombinantly made albumin (such as a
recombinantly made human serum albumin), collagen, gelatin or a
cresol, such as an M-cresol.
[0071] "Stabilizing", "stabilizes", or "stabilization" mean that a
pharmaceutical active ingredient ("PAI") retains at least 20% and
up to 100% of its biological activity (which can be assessed as
potency or as toxicity by an in vivo LD.sub.50 or ED.sub.50
measure) in the presence of a compound which is stabilizing,
stabilizes or which provides stabilization to the PAI. For example,
upon (1) preparation of serial dilutions from a bulk or stock
solution, or (2) upon reconstitution with saline or water of a
lyophilized, or vacuum dried botulinum toxin containing
pharmaceutical composition which has been stored at or below about
-2 degrees C. for between six months and four years, or (3) for an
aqueous solution botulinum toxin containing pharmaceutical
composition which has been stored at between about 2 degrees and
about 8 degrees C. for from six months to four years, the botulinum
toxin present in the reconstituted or aqueous solution
pharmaceutical composition has (in the presence of a compound which
is stabilizing, stabilizes or which provides stabilization to the
PAI) greater than about 20% and up to about 100% of the potency or
toxicity that the biologically active botulinum toxin had prior to
being incorporated into the pharmaceutical composition.
[0072] "Substantially free" means present at a level of less than
one percent by weight of the pharmaceutical composition.
[0073] "Therapeutic formulation" means a formulation can be used to
treat and thereby alleviate a disorder or a disease, such as a
disorder or a disease characterized by hyperactivity (i.e.
spasticity) of a peripheral muscle.
[0074] A pharmaceutical composition within the scope of my
invention can comprise a Clostridial toxin, such as a botulinum
toxin, and an excipient which acts to stabilize the toxin A
pharmaceutical composition within the scope of my invention can
also consist essentially of a botulinum toxin, and a stabilizer.
Additionally, pharmaceutical composition within the scope of my
invention can consist of a botulinum toxin, and a recombinant
stabilizer.
[0075] The botulinum toxin can be present as a botulinum toxin
complex (i.e. as an approximately 300 kiloDalton to about 900
kiloDalton complex, depending upon the particular botulinum toxin
serotype used) or the botulinum toxin can be is present as a pure
or purified botulinum toxin (meaning present as the about 150
kiloDalton neurotoxic component of a botulinum toxin complex) which
is free, substantially or essentially free of any botulinum toxin
complex protein (i.e. removed from association the HA and NTNH
proteins). Thus, as shown by FIG. 1, a botulinum toxin made
naturally by the Clostridium botulinum bacterium is typically made
as a complex comprising the botulinum toxin molecule (a protein
with a molecular weight of about 150 kiloDaltons) (also referred to
as the neurotoxic component) and an array of non-toxic proteins
(hemagglutinins and non-hemagglutinins) in a close, though
non-covalent association with the neurotoxic component. Thus, as
shown by FIG. 1, there can be up to about seven non-neurotoxic
molecules (total weight about 750 kDa) associated (non-covalently)
with the (about 150 kDa) neurotoxic component to form a 900 kDa
botulinum neurotoxin type A complex. In FIG. 1: HA=NTHA=a non-toxic
haemaglutinin; LC=light chain (about 50 kDa); HC=heavy chain (about
100 kDa); --S--S--=the single disulphide bond which joins the LC
and the HC, and; Zn=a zinc atom (botulinum toxin is a zinc
endopeptidase). Hence, LC plus HC is a molecule with a molecular
weight of about 150 kDa, and this is the neurotoxic component of
the (in the case of botulinum toxin type A) 900 kDa complex.
[0076] Any recombinant stabilizer which is present in a
pharmaceutical composition within the scope of my invention can be
a recombinant albumin, a recombinant collagen, a recombinant
gelatin and/or other recombinant primary stabilizer. The
pharmaceutical composition can also comprise a secondary
stabilizer, such as a metal (i.e. zinc) or NAT.
[0077] Significantly, a pharmaceutical composition within the scope
of my invention can have an enhanced potency or stability. By
enhanced potency it is meant that the potency of a first botulinum
toxin pharmaceutical composition is greater than the potency of a
second botulinum toxin pharmaceutical composition. For example a
first botulinum toxin pharmaceutical composition can have a
particular ratio (such as 28:1) of two excipients (such as albumin
and sodium chloride) present in the first composition. A second
botulinum toxin pharmaceutical composition can have a known ratio
(such as about 0.6:1) of the same two excipients (i.e. albumin and
sodium chloride) present in the second composition. Potency and
relative potencies can be determined by a method used to determine
a biological activity of a botulinum toxin, such as a mouse
LD.sub.50 assay. Generally, greater potency means that a lesser
amount (i.e. fewer units) of a botulinum toxin pharmaceutical
composition is required to paralyze a muscle. Preferably, a first
botulinum toxin pharmaceutical composition has at least a 5%
greater potency (and as much as a 50% greater potency) than does
the second botulinum toxin pharmaceutical composition.
[0078] A pharmaceutical composition within the scope of the present
invention can also include a neurotoxin, and a polysaccharide. The
polysaccharide stabilizes the neurotoxin. The pharmaceutical
compositions disclosed herein can have a pH of between about 5 and
7.3 when reconstituted or upon injection.
[0079] The pharmaceutical composition is suitable for
administration to a human patent to achieve a therapeutic effect,
and the neurotoxin can be one of the botulinum toxin serotypes A,
B, C.sub.1, D, E, F and G.
[0080] A further embodiment of the present invention is a method
for using a pharmaceutical composition, the method comprising the
step of local administration of the pharmaceutical composition to a
patient to achieve a therapeutic or cosmetic effect.
[0081] Particular Pharmaceutical Compositions
[0082] My invention encompasses a pharmaceutical composition
comprising: (a) a botulinum toxin; (b) a first excipient, wherein
the first excipient is an albumin, and;
[0083] (c) a second excipient,
[0084] (d) wherein the weight to weight ratio of the first
excipient to the second excipient present in the pharmaceutical
composition is greater than 0.6 and less than about 100. The
potency of the botulinum toxin in this pharmaceutical composition
can be between about 5% greater and about 200% greater than the
potency of a botulinum toxin in a comparison pharmaceutical
composition. The comparison pharmaceutical composition can contain:
(a) the same amount and type of botulinum toxin, and; (b) the same
first and second excipients, as does the pharmaceutical
composition, and; (c) the first and second excipients can be are
present in the comparison pharmaceutical composition in a weight to
weight ratio of 0.6 or less. Additionally, the potency of the
botulinum toxin in the pharmaceutical composition can be between
about 10% greater and about 100% greater than the potency of the
botulinum toxin in the comparison pharmaceutical composition, In
this pharmaceutical composition the botulinum toxin is present as a
botulinum toxin complex or the botulinum toxin can be present as a
pure botulinum toxin (i.e. as a neurotoxic component with a
molecular weight of about 150 kiloDaltons, and at substantially
free of the botulinum toxin complex proteins).
[0085] The first excipient in the pharmaceutical composition can be
a serum albumin or a recombinant albumin. The second excipient in
the pharmaceutical composition can be sodium chloride. The weight
to weight ratio of the first excipient to the second excipient
present in the pharmaceutical composition is between about 1 and
about 50.
[0086] A detailed embodiment of a pharmaceutical composition within
the scope of my invention can comprise: (a) between about
2.0.times.10.sup.-11 grams and about 3.5.times.10.sup.-11 grams of
a botulinum toxin type A for each unit of botulinum toxin present
in the pharmaceutical composition, the unit of the botulinum toxin
being determined by a mouse LD.sub.50 potency assay; (b) an
albumin, and (c) sodium chloride, (d) wherein the weight to weight
ratio of the albumin to the sodium chloride present in the
pharmaceutical composition is greater than 0.6 and less than about
100. The potency of the botulinum toxin in the pharmaceutical
composition can be between about 5% greater and about 200% greater
than the potency of a botulinum toxin in a comparison
pharmaceutical composition, which comparison pharmaceutical
composition contains (a) the same amount and type of botulinum
toxin, and (b) the same albumin and sodium chloride, as does the
pharmaceutical composition of claim 10, and (c) the albumin and
sodium chloride are present in the comparison pharmaceutical
composition in a weight to weight ratio of 0.6 or less. The potency
of the botulinum toxin in the pharmaceutical composition can be
between about 10% greater and about 100% greater than the potency
of the botulinum toxin in the comparison pharmaceutical
composition. Alternately, the weight to weight ratio of the albumin
to the sodium, chloride present in the pharmaceutical composition
can between about 1 and about 50.
[0087] My invention also encompasses a process for making a
pharmaceutical composition by (a) combining about 2.5 ng of a
botulinum toxin type A complex with an albumin and sodium chloride
in a weight to weight ratio of the albumin to the sodium chloride
in the pharmaceutical composition of greater than 0.6 and less than
about 100, to form a mixture, and; (b) vacuum drying the mixture,
to thereby obtain a pharmaceutical composition with a potency after
reconstitution of between about 70 units and about 130 units. This
process can further comprise the step, before the vacuum drying
step, of lyophilizing the mixture. A useful pharmaceutical
composition can be made by this process.
[0088] My invention also encompasses a pharmaceutical composition
suitable for administration to a human, comprising: (a) a botulinum
toxin; (b) sodium chloride, and; (b) a stabilizer, wherein the
potency of the botulinum toxin present in the pharmaceutical
composition is about 40 units/ng. The weight to weight ratio of the
albumin to the sodium chloride present in this pharmaceutical
composition can be greater than 0.6 and less than about 100. The
stabilizer can be a recombinant stabilizer, such as a recombinant
albumin, a recombinant collagen and/or a recombinant gelatin. The
botulinum toxin can be selected from the group consisting of
botulinum toxins types A, B, C, D, E, and F.
[0089] My invention also encompasses a pharmaceutical composition
suitable for administration to a human, comprising: (a) a botulinum
toxin; (b) sodium chloride, and; (b) an albumin, wherein the
potency of the botulinum toxin present in the pharmaceutical
composition is between about 24 units/ng and about 60 units/ng. The
weight to weight ratio of the albumin to the sodium chloride can be
greater than 0.6 and less than about 100.
[0090] My invention also encompasses a pharmaceutical composition
suitable for administration to a human, comprising: (a) a botulinum
toxin; (b) sodium chloride, and;
(b) an albumin, wherein the weight to weight ratio of the albumin
to the sodium chloride present in the pharmaceutical composition is
between about 1 and about 40 and the potency of the botulinum toxin
present in the pharmaceutical composition is between about 24
units/ng and about 60 units/ng.
[0091] My invention also encompasses a process for making a
botulinum toxin pharmaceutical composition which has a potency of
between about 30 to 40 units/ng and is suitable for administration
to a human, the process comprising the steps of: (a) adding a
botulinum toxin type A complex which has a potency of between about
30-40 units/ng to albumin and sodium chloride in a weight to weight
ratio of about 1 to about 40, to form a mixture; (b) vacuum drying
or lyophilizing the mixture, and; (c) reconstituting the mixture
with normal saline, to thereby obtain a botulinum toxin
pharmaceutical composition which has a potency of between about
30-40 units/ng.
[0092] Finally, my invention also encompasses a method for treating
a therapeutic or cosmetic condition, the method comprising the step
of administering to a mammal a botulinum toxin pharmaceutical
composition which has a potency of about 40 units/ng.
[0093] The foregoing methods may be practiced and the compositions
made using a composition that comprises a botulinum toxin type A.
In other embodiments of the invention, the foregoing methods may be
practiced with a composition that comprises botulinum toxin type B.
In further embodiments of the invention, the methods may be
practiced with a composition that comprises a plurality of
botulinum toxin serotypes, such as botulinum toxin serotypes
selected from the group consisting of botulinum toxin serotypes A,
B, C.sub.1, D, E, F and G. In certain embodiments of the invention,
purified botulinum toxins may be used. In other embodiments,
modified botulinum toxins may be used. The compositions used in the
foregoing methods may also include one or more amino acids in
addition to the botulinum toxin and the polysaccharide. Embodiments
of the invention disclosed herein can be administered
intramuscularly (into or to the vicinity of a striated, smooth or
cardiac muscle), intradermally, topically, subcutaneously, into or
to the vicinity of a gland, into a lumen of the body (such as into
a bladder lumen) and/or intrathecally.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] The following drawings illustrate aspects of my
invention.
[0095] FIG. 1 is a diagrammatic representation of a 900 kiloDalton
botulinum toxin complex.
[0096] FIG. 2 is a graph which shows the mouse LD.sub.50 potency
after reconstitution (on the Y-axis) of lyophilized, vacuum-dried
botulinum toxin formulations made with 2.5 ng of botulinum toxin,
500.mu. of HSA and from zero to 10N (9000 .mu.g) of NaCl.
[0097] FIG. 3 presents in three axes recovered potency data for a
number of experimental botulinum toxin formulations. The X axis of
FIG. 3 represents the amount of sodium chloride (NaCl) present in
experimental or research vial preparations in multiples of the
sodium chloride normalized (N) against the 900 .mu.g NaCl content
in a 100 unit vial of Botox.RTM.. Thus, the integer one on the X
axis in FIG. 1 represents 900 .mu.g of NaCl. The Y axis of FIG. 3
represents the amount of human serum albumin (HSA) present in the
same experimental vial preparations in multiples of the HSA
normalized against the 500 .mu.g HSA content in a 100 unit Vial of
Botox.RTM.. Thus, the integer one on the Y axis in FIG. 3
represents 500 .mu.g of HSA. The Z axis of FIG. 3 represents the
recovered potency of these vacuum-dried experimental botulinum
toxin compositions, where each vial contained exactly the same
amount of botulinum toxin (2.5 ng) and where each botulinum toxin
pharmaceutical composition was reconstituted with the same amount
of normal saline, and it's potency after reconstitution determined
by the mouse LD.sub.50 assay.
DETAINED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0098] The present invention is based upon the discovery that a
stable botulinum toxin with an enhanced potency can be made with a
particular ratio of excipients in a botulinum toxin pharmaceutical
composition.
[0099] I have discovered that the potency of a botulinum toxin in a
botulinum toxin pharmaceutical composition can be increased
significantly by formulating the botulinum toxin pharmaceutical
composition with particular ratios of the excipients in the
botulinum toxin pharmaceutical composition. Such particular
excipient ratios permit obtaining a botulinum toxin pharmaceutical
composition which has a higher potency than does a botulinum toxin
pharmaceutical composition with different excipient ratios. For
example, my invention permits a botulinum toxin pharmaceutical
composition to be prepared with a higher potency per nanogram
botulinum toxin present in the botulinum toxin pharmaceutical
composition, than the potency of a botulinum toxin pharmaceutical
composition with different excipient ratios.
[0100] The botulinum toxin present in the botulinum toxin
pharmaceutical composition can be a native, recombinant, hybrid,
chimeric or modified botulinum toxin type A, B, C, D, E, F or G.
Additionally, the botulinum toxin can be present in the botulinum
toxin pharmaceutical composition as either a complex or as a pure
botulinum toxin. A botulinum toxin complex comprises a botulinum
toxin molecule (about 150 kDa) and one or more non-toxic
hemaggluttinin and/or non-toxic non-hemaggluttinin proteins. The
complex can have a molecular weight of, for example, 300, 600 or
900 kDa, with the amount in excess of 150 kDa being attributed to
the non-toxic hemaggluttinin and/or non-toxic non-hemaggluttinin
protein components of the complex. The 150 kDa botulinum toxin
molecule is also referred to as the neurotoxic component and as
pure botulinum toxin.
[0101] An excipient that can be present in a botulinum toxin
pharmaceutical composition can be a protein such as an albumin,
such as a human serum albumin or a recombinantly made albumin.
Another excipient that can be present in a botulinum toxin
pharmaceutical composition can be sodium chloride. Albumin and
sodium chloride can be used as stabilizing excipients in a
botulinum toxin pharmaceutical composition. It is known to use of
sodium chloride and albumin as bulking agents in a botulinum toxin
pharmaceutical composition. The albumin is used as an excipient to
stabilize the toxin during drying and to prevent the toxin from
adhering to surfaces, such as the glass surfaces onto which the
toxin can come into contact during manufacture and storage. See
e.g., Rader R. A., Botulinum toxin A, in Ronald Rader, ed.
BIOPHARMA: Biopharmaceutical Products in the U.S. Market Rockville,
Md.: Biotechnology Information Institute; 2001: pp. 271-274 (332),
and; Rader R. A., Botulinum toxin B, in Ronald Rader, ed.
BIOPHARMA: Biopharmaceutical Products in the U.S. Market Rockville,
Md.: Biotechnology Information Institute; 2001: pp. 274-276
(333).
[0102] An embodiment of my invention is a botulinum toxin
pharmaceutical composition with particular ratios of the albumin to
the sodium chloride present in a botulinum toxin pharmaceutical
compositions. I have found that such ratios permit a significantly
increase in the potency of a reconstituted lyophilized or vacuum
dried botulinum toxin pharmaceutical composition.
[0103] There are a number of reasons why it would not be expected
that altering a ratio of excipients present in a botulinum toxin
pharmaceutical composition would increase the potency of the
botulinum toxin, and why one would expect just the opposite to
occur.
[0104] First, botulinum toxin is a relatively large protein for
incorporation into a pharmaceutical formulation (the molecular
weight of the botulinum toxin type A complex is 900 kD) and is
therefore is inherently fragile and labile. The size of the toxin
complex makes it much more friable and labile than smaller, less
complex proteins, thereby compounding the formulation and handling
difficulties if toxin stability is to be maintained. Hence,
altering a ratio of excipients present in a botulinum toxin
pharmaceutical composition would be expected to denature, fragment
or otherwise detoxify the toxin molecule or cause disassociation of
the non-toxin proteins present in the toxin complex.
[0105] Second, as the most lethal known biological product,
exceptional safety, precision, and accuracy is called for at all
steps of the formulation of a botulinum toxin containing
pharmaceutical composition. Thus, altering a ratio of excipients
present in a botulinum toxin pharmaceutical composition would be
expected to exacerbate or to interfere with the already extremely
stringent botulinum toxin containing pharmaceutical composition
formulation requirements.
[0106] Third, since botulinum toxin was the first microbial toxin
to be approved (by the FDA in 1989) for injection for the treatment
of human disease, specific protocols had to be developed and
approved for the culturing, bulk production, formulation into a
pharmaceutical and use of botulinum toxin. Important considerations
are toxin purity and dose for injection. Hence, altering a ratio of
excipient present in a botulinum toxin pharmaceutical composition
would be expected to interfere with toxin purity and dosage
requirements.
[0107] Fourth, particular difficulties exist to stabilize botulinum
toxin type A, because type A consists of a toxin molecule of about
150 kD in noncovalent association with nontoxin proteins weighing
about 750 kD. The nontoxin proteins are believed to preserve or
help stabilize the secondary and tertiary structures upon which
toxicity is dependant. Procedures or protocols applicable to the
stabilization of nonproteins or to relatively smaller proteins are
not applicable to the problems inherent with stabilization of the
botulinum toxin complexes, such as the 900 kD botulinum toxin type
A complex. Thus while from pH 3.5 to 6.8 the type A toxin and
non-toxin proteins are bound together noncovalently, under slightly
alkaline conditions (pH>7.1) the very labile toxin is released
from the toxin complex. As set forth previously, pure botulinum
toxin (i.e. the 150 kD molecule) has been proposed as the active
ingredient in a pharmaceutical composition. Thus, altering a ratio
of excipient present in a botulinum toxin pharmaceutical
composition would be expected to upset this fragile toxin stability
equilibrium.
[0108] I found that within certain ranges when albumin
concentrations were increased as compared to the sodium chloride
concentration, the potency of the reconstituted botulinum toxin
pharmaceutical composition increased. I also found that within
certain ranges when sodium chloride concentrations were increased
as compared to the albumin concentration, the potency of the
reconstituted botulinum toxin pharmaceutical composition decreased.
Surprisingly, a high (absolute) concentrations of sodium chloride
were found to not be deleterious to potency after reconstitution as
long as a certain ratio of the sodium chloride to the albumin was
maintained. Thus, I discovered that there exist optimal sodium
chloride to albumin ratios (irrespective of the absolute amounts of
sodium chloride or of albumin present) at which an increased
potency of the botulinum toxin pharmaceutical composition after
reconstitution can be obtained.
[0109] As set forth above, I have discovered that establishing
particular sodium chloride to albumin ratios in a botulinum toxin
pharmaceutical composition prior to lyophilization or freeze drying
can be used to optimize the potency of the reconstituted botulinum
toxin pharmaceutical composition.
[0110] Significantly, I also discovered that reconstitution of the
botulinum toxin pharmaceutical composition with normal saline
(0.9%), the typical reconstitution fluid, does not affect the
optimization of the potency botulinum toxin pharmaceutical
composition obtaining by established certain sodium chloride to
albumin ratios in the botulinum toxin pharmaceutical composition
prior to its lyophilization or freeze drying. This discovery
therefore permits conservation of the tonicity of the reconstituted
botulinum toxin pharmaceutical composition administered to a
patient,
[0111] Without wishing to be bound by theory, it can be
hypothesized that establishment of certain sodium chloride to
albumin ratios in the botulinum toxin pharmaceutical composition
prior to its lyophilization or freeze drying permits obtaining an
optimized potency of the botulinum toxin pharmaceutical composition
after it's reconstitution by providing a hospitable chemical and
physical environment for the botulinum toxin during the processing
(compounding or formulation) steps or procedures while also
reducing adsorption of the botulinum toxin upon contact surfaces
(such as glass vials). Presumably, processing of a botulinum toxin
pharmaceutical composition with non-optimized ratios of sodium
chloride and albumin damages the botulinum toxin molecule and
increases it's adsorption to surfaces.
[0112] Thus, changing the NaCl/HSA ratio to a particular ratio may
increase toxin potency because less toxin is thereafter lost during
freezing to denaturation and adsorption to surfaces (these two
phenomenon may be related, e.g., denaturation caused by
adsorption). HSA can act as a cryoprotectant and NaCl as a
degradant. Typically, some amount of a botulinum toxin will adhere
to glass surface. I have determined that when less NaCl is present
in a botulinum toxin pharmaceutical composition, the vacuum-dried
toxin does not adhere to a glass surface, thereby indicating that
NaCl can facilitate or cause adsorption of the toxin to processing
or storage surfaces. Thus particular HSA:NaCl ratios can be optimal
because they permit the HSA to provide sufficient cryoprotection
while at the same time counteracting a deleterious effect (surface
adsorption) caused by of the NaCl present.
[0113] Thus, my discovery permits a botulinum toxin pharmaceutical
composition to be made with a potency that is essentially 100% of
the theoretically possible potency. Additionally the optimized
potency is maintained after reconstitution with saline, which
permits administration of an isotonic botulinum toxin
pharmaceutical composition. The result is a more efficient
manufacturing use of the botulinum toxin active ingredient and a
reduced patient exposure to degraded botulinum toxin in the final
botulinum toxin pharmaceutical composition.
[0114] A significant advantage of my invention is that it permits a
botulinum toxin pharmaceutical composition to be manufactured
(compounded from a raw or bulk botulinum toxin) with considerably
less botulinum toxin. This permits a more efficient manufacturing
process in which less bulk toxin is used to make the final
botulinum toxin pharmaceutical composition. Additionally, because
the final botulinum toxin pharmaceutical composition contains less
botulinum toxin, the patient, on a unit to unit basis, is
administered less botulinum toxin with the ensuing advantages of
fewer side effects, such as reduced immunogenicity. Specifically,
my invention permits a botulinum toxin pharmaceutical composition
to be made with the same potency but with from about 5% less to
about 50% less total botulinum toxin present in the botulinum toxin
pharmaceutical composition. For example, a botulinum toxin
pharmaceutical composition which formerly comprised about 20 units
of botulinum toxin for each 1 ng of botulinum toxin present can,
according to my invention, now be made with as much as 40 units of
botulinum toxin for each 1 ng of botulinum toxin present in the
botulinum toxin pharmaceutical composition.
[0115] A botulinum toxin pharmaceutical composition manufacturing
(compounding) process can typically require as much as a 50%
overage, meaning that the manufacturing process which is initiated
with a 1.5.times. amount (i.e. 150 units) of a botulinum toxin
provides a botulinum toxin pharmaceutical composition with an
amount 1.times. (i.e. 100 units) of the botulinum toxin. A
manufacturing or compounding process is the process by which a
botulinum toxin (referred to as bulk or raw toxin) obtained from
bacterial fermentation is then diluted, compounded and processed
for the preparation of a botulinum toxin pharmaceutical composition
suitable for administration to humans for therapeutic and/or
cosmetic purposes.
[0116] Thus, there is an unexplained loss of up to 50% of the
potency of the botulinum toxin during the compounding process. It
has been postulated that the 50% overage is required due to
denaturation and/or loss of botulinum toxin during the compounding
process. My invention indicates that the up to 50% overage of
botulinum toxin is not lost during compounding, but rather the
chemical composition of the formulation is the critical parameter
in achieving full recovery of toxin in the finished product. No
overage is necessary when optimized ratios of the excipients are
used by providing the appropriate environment during the
vacuum-drying process. While not wanting to be bound by theory, it
is plausible that optimal ratios reduce adsorption to surfaces and
denaturation during the drying process.
[0117] Thus, my invention permits a botulinum toxin pharmaceutical
composition to be made with eg from about 34% less (if the amount
of botulinum toxin in a 100 unit vial is reduced from 3.8 ng to 2.5
ng) to about 48% less (if the amount of toxin in a 100 unit vial is
reduced from 4.8 ng to 2.5 ng, to about to about 50% less (if the
amount of toxin in a 100 unit vial is reduced from 5.0 ng to 2.5
ng.
[0118] Human serum albumin (plasma derived) is available
commercially from various sources, including, for example, from
Bayer Corporation, pharmaceutical division, Elkhart, Ill., under
the trade name Plasbumin.RTM.. Plasbumin.RTM. is known to contain
albumin obtained from pooled human venous plasma as well as sodium
caprylate (a fatty acid, also known as octanoate) and
acetyltryptophan ("NAT"). See e.g. the Bayer Plasbumin.RTM.-20
product insert (directions for use) supplied with the product and
as published at http://actsysmedical.com/PDF/plasbumin20.pdf. The
caprylate and acetyltryptophan in commercially available human
serum albumin are apparently added by FDA requirement to stabilize
the albumin during pasteurization at 60 degrees C. for 10 hours
prior to commercial sale. See e.g. Peters, T., Jr., All About
Albumin Biochemistry, Genetics and Medical Applications, Academic
Press (1996), pages 295 and 298. Recombinant human albumin is
available from various sources, including for example, from Bipha
Corporation of Chitose, Hokkaido, Japan, Welfide Corporation of
Osaka, Japan, and from Delta Biotechnology, Nottingham, U.K., as a
yeast fermentation product, under the trade name
Recombumin.RTM..
[0119] It is known to express recombinant human serum albumin
(rHSA) in the yeast species Pichia pastoris. See e.g. Kobayashi K.,
et al., The development of recombinant human serum albumin, Ther
Apher 1998 November; 2(4):257-62, and; Ohtani W., et al.,
Physicochemical and immunochemical properties of recombinant human
serum albumin from Pichia pastoris, Anal Biochem 1998 Feb. 1;
256(1):56-62. See also U.S. Pat. No. 6,034,221 and European patents
330 451 and 361 991. A clear advantage of a rHSA is that it is free
of blood derived pathogens.
[0120] The excipient ratios set forth herein can it is believed
help to provide stability to a neurotoxin active ingredient, such
as a botulinum toxin, present in the pharmaceutical composition by:
(1) reducing adhesion (commonly referred to as "stickiness") of the
botulinum toxin to surfaces, such as the surfaces of laboratory
glassware, vessels, the vial in which the pharmaceutical
composition is reconstituted and the inside surface of the syringe
used to inject the pharmaceutical composition. Adhesion of the
botulinum toxin to surfaces can lead to loss of botulinum toxin and
to denaturation of retained botulinum toxin, both of which reduce
the toxicity of the botulinum toxin present in the pharmaceutical
composition; (2) reducing the denaturation of the botulinum toxin
and/or dissociation of the botulinum toxin from other non-toxin
proteins present in the botulinum toxin complex, which denaturation
and/or dissociation activities can occur because of the low
dilution of the botulinum toxin present in the pharmaceutical
composition (i.e. prior to lyophilization or vacuum drying) and in
the reconstituted pharmaceutical composition; (3) reducing loss of
botulinum toxin (i.e. due to denaturation or dissociation from
non-toxin proteins in the complex) during the considerable pH and
concentration changes which take place during preparation,
processing and reconstitution of the pharmaceutical
composition.
[0121] The three types of botulinum toxin stabilizations presumably
provided by the ratios set forth herein can conserve and preserve
the botulinum toxin with it native toxicity prior to injection of
the pharmaceutical composition.
[0122] In certain embodiments of the invention, the pharmaceutical
compositions of the invention may comprise a plurality of botulinum
toxin serotypes. In other words, the composition may include two or
more different botulinum toxin serotypes. For example, a
composition may include botulinum toxin serotypes A and B. In
another embodiment, a composition may include botulinum toxin
serotypes A and E. Using a combination of botulinum toxin serotypes
will permit caregivers to customize the composition to achieve a
desired effect based on the condition being treated. In an
additional embodiment of the invention, the composition may
comprise a modified botulinum toxin. The modified botulinum toxin
will preferably inhibit the release of neurotransmitter from a
neuron, but may have a greater or lower potency than the native
botulinum toxin, or may have a greater or lower biological effect
than the native botulinum toxin. Because the compositions of the
invention may be used for relatively long-term treatment of
animals, the compositions may be provided in a relatively pure
form. In one embodiment, the compositions are of a pharmaceutical
grade. In certain embodiments, the clostridial neurotoxin has a
greater than 95% purity. In additional embodiments, the clostridial
neurotoxin has a purity greater than 99%.
[0123] My invention also encompasses addition of a preservative,
either in the diluent or formulation itself, to allow extended
storage. A preferred preservative is preserved saline containing
benzyl alcohol.
[0124] A liquid formulation can be advantageous. A single-step
presentation (e.g., pre-filled syringe) or a product configuration
that the user perceives as a single-step presentation (e.g.,
dual-chambered syringe) would provide convenience by eliminating
the reconstitution step. Freeze-drying is a complicated, expensive
and difficult process. Liquid formulations are often easier and
cheaper to produce. On the other hand liquid formulations are
dynamic systems and therefore more susceptible to excipient
interaction, fast reactions, bacterial growth, and oxidation than
freeze-dried formulations. A compatible preservative might be
needed. Anti-oxidants such as methionine might also be useful as
scavengers especially if surfactants are used to reduce adsorption
as many of these compounds contain or produce peroxides. Any of the
stabilizing excipients which can be used in a freeze-dried
formulation (e.g., hydroxyethyl starch or an amino acid such,
lysine) might be adapted to use in a liquid formulation to assist
in reducing adsorption and stabilize the toxin. Suspensions similar
to those developed for insulin are also good candidates.
Additionally, stabilizing botulinum toxin in a liquid vehicle might
require a low pH vehicle as the toxin is reported to be labile
above pH 7. This acidity could produce burning and stinging upon
injection. A binary syringe could be employed. Inclusion of a
co-dispensed buffer, sufficient to raise the pH to physiologic
levels, would alleviate injection discomfort of a low pH while
maintaining the toxin at a low pH during storage. Another
dual-chambered syringe option would include diluent and lyophilized
material segregated in separate chamber, only mixing upon use. This
option provides the advantages of a liquid formulation without the
additional resources and time.
[0125] As discussed herein, the neurotoxin may be prepared and
purified using techniques well-known in the art. The purified toxin
may subsequently be diluted in a stabilizer such as a
polysaccharide (e.g., hetastarch), or a recombinant serum albumin,
or a serum albumin of the species of animal receiving the
neurotoxin. It is preferred that the stabilizer prevents or reduces
denaturation of the toxin, and produces no, or minimal, immunogenic
responses in the animal that will receive the toxin. Aliquots of
the diluted toxin are then lyophilized using conventional
procedures.
[0126] The lyophilized neurotoxin may be reconstituted before
administering the neurotoxin to a subject by adding water, saline,
or any buffer solution to the lyophilized neurotoxin. In certain
embodiments, sodium free buffers may be preferred to help reduce
denaturation of the neurotoxin.
[0127] The pharmaceutical compositions of the invention can be
administered using conventional modes of administration. In
preferred embodiments of the invention, the compositions are
administered intramuscularly or subcutaneously to the subject. In
other embodiments, the compositions of the invention may be
administered intrathecally. In addition, the compositions of the
invention may be administered with one or more analgesic or
anesthetic agents.
[0128] The most effective mode of administration and dosage regimen
for the compositions of this invention depends upon the type,
severity, and course of the condition being treated, the animal's
health and response to treatment, and the judgment of the treating
doctor. Accordingly, the methods and dosages of the compositions
should be tailored to the individual subject.
[0129] The compositions of the invention may also be injected into
smooth muscles (as compared to striated muscles) to treat colonic,
bladder, esophageal, or gastrointestinal dysfunction, including,
but not limited to achalasia, anal fissure, hyperactive sphincter
of oddi. The administration of the compositions may reduce or
prevent unfavorable systemic consequences from treatment with drugs
that do not specifically act on the organ of interest.
[0130] Compositions containing botulinum toxin may be administered
intramuscularly, intrathecally, or subcutaneously to relieve pain
experienced by the animal. These treatments are also restricted to
the site of injection and have minimal side effects compared to
current systemic approaches of treating these pain syndromes with
pain relieving drugs.
[0131] Relief from pain by practicing the methods of the invention
may be determined by observing the reduction in the number of
symptoms that the animal is exhibiting. One or more of the symptoms
may be reduced.
[0132] As indicated above, dosages of the neurotoxin, such as
botulinum toxin, in the compositions may vary. In one embodiment,
the compositions contain a therapeutically effective amount of
neurotoxin, for example, between about 1 U and about 500 U of
botulinum toxin type A. Preferably the amounts are between about 10
U and about 300 U. More preferably the amount is between about 20 U
and 250 U, such about 50 U to 200 U, or 70 U.
[0133] Alternatively, botulinum toxin, such as botulinum toxin type
A, can be administered in amounts between about 10.sup.-3 U/kg and
about 60 U/kg to alleviate pain experienced by a mammal.
Preferably, the botulinum toxin used is administered in an amount
of between about 10.sup.-2 U/kg and about 50 U/kg. More preferably,
the botulinum toxin is administered in an amount of between about
10.sup.-1 U/kg and about 40 U/kg. Most preferably, the botulinum
toxin is administered in an amount of between about 1 U/kg and
about 30 U/kg. In a particularly preferred embodiment of the
present disclosed methods, the botulinum toxin is administered in
an amount of between about 1 U/kg and about 20 U/kg.
[0134] Compositions containing other serotypes of botulinum toxin
may contain different dosages of the botulinum toxin. For example,
botulinum toxin type B may be provided in a composition at a
greater dose than a composition containing botulinum toxin type A.
In one embodiment of the invention, botulinum toxin type B may be
administered in an amount between about 1 U/kg and 150 U/kg.
Botulinum toxin type B may also be administered in amounts of up to
20,000 U (mouse units, as described above). In another embodiment
of the invention, botulinum toxin types E or F may be administered
at concentrations between about 0.1 U/kg and 150 U/kg. In addition,
in compositions containing more than one type of botulinum toxin,
each type of botulinum toxin can be provided in a relatively
smaller dose than the dose typically used for a single botulinum
toxin serotype. The combination of botulinum toxin serotypes may
then provide a suitable degree and duration of paralysis without an
increase in diffusion of the neurotoxins (e.g. see U.S. Pat. No.
6,087,327).
[0135] The compounding process used in the preparation of
Botox.RTM. (a vacuum dried powdered pharmaceutical composition)
begins with the entry of about 150 units of the bulk botulinum
toxin into the compounding process. The final product (ready for
reconstitution) comprises only about 100 units of the botulinum
toxin (as well as specific amount of albumin and sodium chloride as
excipients added during the compounding process). Thus, about 50
units of botulinum toxin (about one third of the amount of the
botulinum toxin which entered into the compounding process) is lost
during compounding, for example by denaturation, absorption and
inactivation of the botulinum toxin onto processing surfaces.
Hence, it has been necessary to start the compounding process with
about a 50% overage (i.e. start with about 150 units of botulinum
toxin in order to obtain product with about 100 units of botulinum
toxin).
[0136] One hundred units of botulinum toxin typically comprises
from about 3.6 ng to about 5 ng of botulinum toxin. I have
discovered that by altering the ratio of the albumin and sodium
chloride excipients used in the compounding process it is possible
to: (1) remove the 50% overage factor, and; (2) on a nanogram to
nanogram basis, obtain a more potent botulinum toxin. By
eliminating the 50% overage factor in the compounding process, one
can have about 100 units of botulinum toxin enter the compounding
process and yet at the completion of the compounding process still
obtain product with about 100 units of botulinum toxin.
Additionally, one can obtain a 100 botulinum toxin unit product
comprising only about 2.4 ng of botulinum toxin, as compared to the
previously required 3.6 to 5 ng of botulinum toxin required to
obtain a 100 unit botulinum toxin product. Thus, the potency of the
botulinum toxin which was previously as low as about 20 units per
ng (100 units divided by 5 ng) can now be increased to as high as
about 42 units per ng (100 units divided by 2.4 ng), which equates
to an increase of potency of about 110%.
[0137] Commercially available botulinum toxin pharmaceutical
compositions approved by regulatory agencies for use in humans to
treat one or more indications include BOTOX.RTM. (Allergan, Inc,
Irvine, Calif.), Dysport.RTM. (Ipsen Pharmaceuticals, Paris,
France) and MyoBloc.TM. (Solstice Neurosciences, San Diego,
Calif.).
EXAMPLES
[0138] The following non-limiting examples provide those of
ordinary skill in the art with specific preferred formulations and
methods and are not intended to limit the scope of the
invention.
[0139] In the Examples below the well known mouse lethal
dose.sub.50 assay (the "MLD50") was used to determine recovered
potency of the botulinum toxin formulations made. The MLD50 is a
method for measuring the potency of a botulinum toxin by
intraperitoneal injection of the botulinum toxin into female mice
(about four weeks old) weighing 17-22 grams each at the start of
the assay. Each mouse is held in a supine position with its head
tilted down and is injected intraperitoneally into the lower right
abdomen at an angle of about 30 degrees using a 25 to 27 gauge
3/8'' to 5/8'' needle with one of several serial dilutions of the
botulinum toxin in saline. The death rates over the ensuing 72
hours for each dilution are recorded. The dilutions are prepared so
that the most concentrated dilution produces a death rate of at
least 80% of the mice injected, and the least concentration
dilution produces a death rate no greater than 20% of the mice
injected. There must be a minimum of four dilutions that fall
within the monotone decreasing range of the death rates. The
monotone decreasing range commences with a death rate of no less
than 80%. Within the four or more monotone decreasing rates, the
two largest and the two smallest rates must be decreasing (i.e. not
equivalent). The dilution at which 50% of the mice die within the
three day post injection observation period is defined as a
dilution which comprises one unit (1 U) of the botulinum toxin.
[0140] In the Tables which follow "normalized" means with regard to
the amount used in a 100 unit vial of Botox.RTM..
Example 1
High Potency Botulinum Toxin Formulations (Research Method) with
Various Ratios of Sodium Chloride to Albumin
[0141] An experiment was carried out to assess the recovered
potency of numerous botulinum toxin research vial formulations with
the same amount of botulinum toxin type A complex in each
formulation, but with different amounts of HSA and NaCl present in
the each formulation. Thus, while 2.5 ng of the botulinum toxin was
used consistently per vial, each formulation vial contained from 0N
(0 .mu.g) of HSA to 10N (5000 .mu.g) of HSA, and from 0N (0 .mu.g)
of NaCl to 10N (9000 .mu.g) of NaCl.
[0142] The data in Table 1 was obtained using research lot
preparation procedures. Thus, the Table 1 data was obtained by
mixing one of the specified seven different amounts of sodium
chloride (NaCl) (from a 0.1N amount of 90 .mu.g per vial to a 10N
amount of 9000 .mu.g per vial) with one of the specified three
different amounts of human serum albumin (HSA) (Bayer) (from a 0.5N
amount of 250 .mu.g per vial to a 1N amount of 500 .mu.g per vial)
with sterile water and the same amount (2.5 ng/vial) of botulinum
toxin type A complex so as to prepare experimental or research
botulinum toxin pharmaceutical composition vials. These vials were
then placed in a lyophilizer, vacuum-dried and stoppered. The vials
were then reconstituted with normal saline and tested for recovered
potency using a refined version of the mouse potency LD.sub.50
assay.
[0143] Significantly, Table 1 shows that, as compared to the
Botox.RTM. equivalent research formulation (that is, the research
vial formulation which contained 500 .mu.g of HSA, 900 .mu.g of
NaCl and 2.5 ng of botulinum toxin type A complex, and had a
recovered potency of 57 units), a high potency (61 to 89 units of
botulinum toxin activity per vial, that is from 107% to 156%
greater than the potency of the Botox.RTM. equivalent research
formulation) was obtained for each of the six formulations which
had HSA:NaCl ratios greater than 0.6 (the HSA: NaCl ratios in Table
1 that were greater than 0.6 were ratios from 0.7 to 5.6.
[0144] The Table 1 data therefore shows that a high potency
botulinum toxin formulation can be obtained by increasing the
HSA:NaCl ratio of the formulation.
TABLE-US-00001 TABLE 1 Potency of Botulinum Toxin Formulations with
HSA:NaCl Ratios from 0.03 to 5.6 Normalized NaCl Normalized HSA
(.mu.g/vial) (.mu.g/vial) 0.5 (250) 0.75 (375) 1 (500) 0.1 (90) 81
(2.8) 74 (4.2) 89 (5.6) 0.5 (450) 49 (0.6) 68 (0.8) 64 (1.1) 0.75
(675) 45 (0.4) 60 (0.6) 61 (0.7) 1 (900) 44 (0.3) 52 (0.4) 57 (0.6)
2 (1800) 49 (0.1) 56 (0.2) 62 (0.3) 5 (4500) 41 (0.06) 46 (0.08) 59
(0.1) 10 (9000) 49 (0.03) 48 (0.04) 58 (0.06)
[0145] A further example showing the potency of reconstituted
research formulations made according to the procedures set forth in
this Example 1 is provided by Table 2 below and by FIG. 2. Table 2
shows that formulations which contained a constant 1N (500 .mu.g)
amount of HSA, 2.5 ng of botulinum toxin and amounts of NaCl which
varied from zero to 10N (9000 .mu.g) had a potency which did not
vary significantly as the HSA:NaCl ratio was decreased below 0.6,
but that the potency of the formulation increased from about 57
units to about 90 units, as the HSA:NaCl ratio was increased from
0.6 to about 5.6.
TABLE-US-00002 TABLE 2 Potency of Botulinum Toxin Formulations with
1N and HSA:NaCl Ratios from 0.06 to 5.6 Normalized Amount of NaCl
HSA:NaCl ratio Potency 10 0.06 59 5 0.1 60 2 0.3 61 1 0.6 58 0.75
0.7 62 0.5 1.1 65 0.1 5.6 89
[0146] FIG. 2 is a graphical representation of the Table 2
data.
[0147] FIG. 3 presents a combined view in three axes of the data
generated by this Example 1. FIG. 3 shows that as the HSA:NaCl
ratio is increased beyond about 0.6 (by either: (a) holding the
amount of HSA in the formulation constant [at an amount between 0
and 10N] and decreasing the amount of NaCl in the formulation [from
10N to 0], or by; (b) holding the amount of NaCl constant and
increasing the amount of HSA in the formulation), generally the
recovered potency of the formulation increases. The amount of
botulinum toxin in all the formulations used to generate the FIG. 3
data was constant at 2.5 ng per vial.
Example 2
High Potency Botulinum Toxin Formulations (Commercial Method) with
Particular Ratio of Sodium Chloride to Albumin
[0148] A further experiment was carried out in which botulinum
toxin pharmaceutical compositions were made (compounded) using
botulinum toxin type A complex, sodium chloride and human serum
albumin. Botulinum toxin pharmaceutical compositions containing
differing ratios of sodium chloride to the HSA were compounded
using commercial manufacturing lot procedures. The compositions
were then either lyophilized and vacuum dried to a solid, powder)
state, followed by reconstitution with saline and mouse LD.sub.50
recovered potency evaluation.
[0149] Results obtained are set forth in Table 3. The Table 3 data
was obtained as follows: for the BOTOX data (last row in Table 3)
100 units vials of Botox.RTM. were reconstituted with normal saline
followed by use of the mouse LD.sub.50 assay to measure potency.
The 1N HSA, 2N HSA, 5N HSA and 10N HSA represent formulations
compounded in the same way used for the manufacture of Botox.RTM.
and with the same excipients, reconstituted in the same manner, and
potency assessed using the same mouse LD.sub.50 assay, with only
the following changes:
[0150] (1) one (1N), two (2N), five (5N) or ten times (10N) as much
HSA was used in the compounding process for the manufacture of
these four botulinum toxin type A complex formulations, as compared
to how much HSA is used in the manufacture of Botox.RTM.. Thus, as
shown in Table 3, the 1N (1N meaning normalized to contain the same
amount of that excipient as is present in a 100 unit vial of
Botox.RTM.) formulation was compounded to contain 500 .mu.g of HSA,
while the 10N formulation was compounded to contain 5000 .mu.g of
HSA.
[0151] (2) the amount of botulinum toxin type A complex used in
each of the four 1N HSA, 2N HSA, 5N HSA and 10N HSA formulations
was 2.4 ng.
[0152] (3) the amount of sodium chloride used in the compounding
process for the manufacture of each of the four botulinum toxin
type A complex formulations was altered so as to provide, a
constant weight to weight ratio the HSA to the NaCl in the
formulation of 28. Thus, in each of these four formulations the
HSA:NaCl ratio was 47 times what it is for Botox.RTM. (28 vs
0.6).
[0153] Significantly, Table 3 shows that a high potency (95 to 103
units of botulinum toxin activity per vial) was obtained for each
of the four HSA:NaCl ratio of 28 botulinum toxin formulations, and
that this high potency was obtained upon use of from about 33% to
about 52% less botulinum toxin (as compared to use of from about
3.6 ng to about 5 ng of botulinum toxin per vial of
Botox.RTM.).
[0154] Additionally, as shown by Table 3, through wide ranges of
the absolute amounts of HSA and sodium chloride present, a weight
to weight ratio of HSA to sodium chloride of 28 consistently
permitted a potency of about 40 units per ng of the compounded and
reconstituted botulinum toxin to be obtained.
TABLE-US-00003 TABLE 3 Potency of Botulinum Toxin Formulations with
an HSA:NaCl ratio of 28 HSA:NaCl Toxin NaCl HSA Potency Wt. Ratio
(ng/vial) (.mu.g/vial) (.mu.g/vial) (n = 2) 1N HSA 28 2.4 18 500 96
2N HSA 28 2.4 36 1000 103 5N HSA 28 2.4 90 2500 98 10N HSA 28 2.4
180 5000 95 BOTOX 0.6 3.6 to 5 900 500 70-100
[0155] Thus, this experiment showed that the manufacturing
(compounding) process 50% overage can be eliminated by altering the
composition ratio of the formulation while still using existing
manufacturing processes and excipients.
[0156] A pharmaceutical composition according to the invention
disclosed herein has many advantages, including that the
pharmaceutical composition can have high stability and high %
recovery of toxin potency comparable to or superior to that
achieved with currently available pharmaceutical compositions.
[0157] Various publications and/or references have been cited
herein, the contents of which, in their entireties, are
incorporated herein by reference.
[0158] Although the present invention has been described in detail
with regard to certain preferred methods, other embodiments,
versions, and modifications within the scope of the present
invention are possible. For example, a wide variety of stabilizing
polysaccharides, proteins and amino acids are within the scope of
the present invention.
[0159] Accordingly, the spirit and scope of the following claims
should not be limited to the descriptions of the preferred
embodiments set forth above.
* * * * *
References