U.S. patent application number 15/422083 was filed with the patent office on 2017-05-25 for non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof.
The applicant listed for this patent is MEDEXGEN INCORPORATED. Invention is credited to Yong Hoon CHUNG, Hyun Sub LEE.
Application Number | 20170145399 15/422083 |
Document ID | / |
Family ID | 45067165 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170145399 |
Kind Code |
A1 |
CHUNG; Yong Hoon ; et
al. |
May 25, 2017 |
NON-DIFFUSIVE BOTULINUM TOXIN CAUSING LOCAL MUSCLE PARALYSIS, AND
PURIFICATION METHOD THEREOF
Abstract
The present invention relates to a method for purifying a
non-spreading botulinum toxin that causes local muscle paralysis
and a non-spreading botulinum toxin obtained thereby. The method
comprises the steps of: subjecting a purified botulinum toxin type
A product to ion-exchange chromatography using a controlled pH of
buffer, concentration of sodium chloride (NaCl), thereby separating
the botulinum toxin type A product into subtractions; and
collecting a subfraction having an A260/A280 value in a specific
range from the separated subfractions.
Inventors: |
CHUNG; Yong Hoon; (Seoul,
KR) ; LEE; Hyun Sub; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDEXGEN INCORPORATED |
Seoul |
|
KR |
|
|
Family ID: |
45067165 |
Appl. No.: |
15/422083 |
Filed: |
February 1, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13700867 |
Nov 29, 2012 |
9598683 |
|
|
PCT/KR2011/003547 |
May 13, 2011 |
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15422083 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/00 20180101;
C12Y 304/24069 20130101; A61K 38/4893 20130101; A61P 23/02
20180101; A61P 21/00 20180101; C12N 9/52 20130101; G01N 30/96
20130101; A61K 49/00 20130101; A61K 49/0004 20130101; A61K 38/00
20130101 |
International
Class: |
C12N 9/52 20060101
C12N009/52 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2010 |
KR |
10-2010-0051076 |
Claims
1. A non-spreading botulinum toxin, comprising Zn, Fe, and Mg ion
concentrations at least 150, 80, and 140 ppb per 100 U/ml,
respectively.
2. The non-spreading botulinum toxin of claim 1, wherein the
non-spreading botulinum toxin comprises Zn, Fe, and Mg ion
concentrations at least 300, 100, and 180 ppb per 100 U/ml,
respectively.
3. A non-spreading botulinum toxin prepared by a process comprising
the steps of: purifying a botulinum toxin type A by an acid
precipitation; separating subfractions from the purified botulinum
toxin type A by conducting anion-exchange chromatography having 1
ml column volume, wherein: the anion-exchange chromatography is
conducted with a condition of pH 4.5-6.5 of sodium acetate as
buffer and 0-1 M of sodium chloride (NaCl), a loading volume of the
purified botulinum toxin type A conducted by the anion-exchange
chromatography ranges 0.25-2 ml, the subfractions contain a peak I,
a peak II, and a peak III, and the peak I, peak II, and peak III
are obtained with 0 M, 0.2-0.2 M, and 1 M of the sodium chloride
(NaCl), respectively; and selecting a non-spreading botulinum toxin
having an A260/A280 value of 0.45-0.59 from the peak I, peak II,
and peak III.
4. The non-spreading botulinum toxin of claim 3, wherein the
non-spreading botulinum toxin comprises Zn, Fe, and Mg ion
concentrations at least 150, 80, and 140 ppb per 100 U/ml,
respectively.
5. The non-spreading botulinum toxin of claim 4, wherein the
non-spreading botulinum toxin comprises Zn, Fe, and Mg ion
concentrations at least 300, 100, and 180 ppb per 100 U/ml,
respectively.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for purifying a
non-spreading botulinum toxin that causes local muscle paralysis
and, a non-spreading botulinum toxin obtained thereby.
BACKGROUND ART
[0002] The US Food and Drug Administration (FDA) warned that
Botox.RTM. which is used for cosmetic purposes such as wrinkle
removal can cause serious side effects, such as death, in severe
cases.
[0003] The US FDA said that a side effect of muscle paralysis in
areas other than an area injected with Botox was reported and
persons injected with Botox.RTM. were hospitalized or died.
[0004] In addition, the US FDA said that patients in which serious
Botox.RTM. side effects occurred were mostly child patients whose
leg muscles were injected with Botox.RTM. in order to treat
muscular stiffness caused by cerebral palsy.
[0005] Botox.RTM. is widely used for the treatment of the muscles
of cerebral palsy patients, neck muscle stiffness, pain caused by
muscular stiffness, and vocal cord palsy. In the field of cosmetic
therapy, Botox.RTM. is used for the removal of wrinkles and the
prevention or treatment of aging. As anti-aging therapies have
recently been popularized, the amount of Botox.RTM. used has
increased annually.
[0006] When Botox.RTM. is injected into muscles, the muscular
nerves are paralyzed. Thus, Botox.RTM. has been injected into
children with cerebral palsy whose leg muscles are excessively
stiff, in order to make walking more natural.
[0007] The FDA considers that Botox.RTM. influences respiratory
muscle function while spreading to areas other than legs, thereby
causing side effects [FDA NEWS RELEASE FOR IMMEDIATE RELEASE Apr.
30, 2009, FDA Patient Safety News: Show #74, April 2008]. In
connection with this, the FDA said that patients and doctors need
to pay attention to whether a decrease in breathing rate or
difficulty in swallowing occurs after injection with Botox.RTM.
[FDA-approved Patient Labeling Jul. 31, 2009 APPENDIX 1: MEDICATION
GUIDE BOTOX, BOTOX Cosmetic (Boe-cox.RTM.) (on a botulinum toxin A)
for Injection)].
[0008] However, the FDA said that a serious side effect of
Botox.RTM. injection for cosmetic purposes such as wrinkle removal
has not yet been reported. The FDA did not require doctors to stop
Botox.RTM. treatments for cosmetic purposes.
[0009] The US consumers union asked the FDA to strengthen the
warning that 180 cases of Botox.RTM.-related side effects were
reported to the FDA between the years 1997 and 2006 and 16 cases
thereof led to death and that the use of Botox.RTM. can cause
abnormalities.
[0010] In prior art, botulinum toxin has been purified by
performing acid precipitation (U.S. Pat. No. 7,354,740, entitled
"Animal product free system and process for purifying a botulinum
toxin", Allergan, Inc.) or performing chromatography following acid
precipitation (U.S. Pat. No. 7,452,697, entitled "Chromatographic
method and system for purifying a botulinum toxin", Allergan, Inc;
Korean Patent Application No. 10-2008-0016800, entitled "Method of
purifying botulinum toxin from Clostridium botulinum, Medexgen
Inc.; Korean Patent Application No. 10-2002-0000685, entitled,
"Method for purifying Clostridium botulinum type A toxin", Medexgen
Inc.).
[0011] However, it is known that the botulinum toxins purified by
the above methods significantly spread, to areas in the body other
than the injected area to paralyze the surrounding organs or
respiratory muscles, and in severe cases, cause serious side
effects leading to death.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0012] Therefore, the present invention has been made in view of
the above-mentioned problems, and the present inventors have found
that, when an existing botulinum toxin type A purified by either
acid precipitation or chromatography following acid precipitation
is subjected to ion-exchange chromatography using sodium chloride,
three, heterogeneous subfractions are obtained, and one
sub-fraction thereof has muscle-paralyzing activity and, at the
same time, does not spread in the body.
[0013] Therefore, it is an object of the present invention to
provide a non-spreading botulinum toxin, which does not spread in
the body and, at the same time, localize muscle-paralyzing
activity, and a method for purifying the same.
Technical Solution
[0014] To achieve the above objects, in accordance with one aspect
of the present invention, there, is provided a method for purifying
a non-spreading botulinum toxin, comprising the steps of:
separating the botulinum toxin type A product into subfractions by
conducting ion-exchange chromatography using pH 4.5-6.5 buffer and
0.02-0.2 M of sodium chloride (NaCl); and collecting a
non-spreading botulinum toxin subfraction, which has an A260/A280
value of 0.4-0.6.
[0015] In accordance with still another aspect of the present
invention, there is provided a non-spreading botulinum toxin
preparation, which is purified by the above method comprises, Zn,
Fe and Mg ion concentrations at least 150, 80, and 140 ppb per 100
U/ml, respectively.
[0016] In accordance with still another aspect of the present
invention, there is provided a method for determining a
non-spreading botulinum toxin, comprising: injecting the
non-spreading botulinum toxin into the either left or right hind
limb calf muscle of mouse (4-6 wk old, weighing 18-22 g) in an
amount equivalent to 1.5-3 times the LD.sub.50 of the toxin; and
determining whether the right hind limb muscles and respiratory
muscles of the mouse were paralyzed and whether the mouse died.
[0017] In accordance with still another aspect of the present
invention, there is provided the said method, wherein the
non-spreading botulinum toxin preparation shows a survival rate at
least 80% at 96 hours after injection of 2 U toxin preparation in
the volume 50 .mu.L either left or right hind limb calf muscle of
mouse (4-6 wk old, weighing 18-22 g).
Advantageous Effects
[0018] According to the present invention, a non-spreading
botulinum toxin that causes local muscle paralysis can be obtained
which causes muscle paralysis in a desired area, does not spread to
other areas from administration site and has the effect of
paralyzing muscles in a fast and lasting manner.
[0019] Also, it is possible to obtain a non-spreading botulinum
toxin having a high toxin titer compared to conventional botulinum
toxin products.
[0020] In addition, according to the present invention, a
non-spreading botulinum toxin which is not mixed with a spreading
toxin can be obtained in large amounts by controlling anion
chromatographic conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 shows the results of separating botulinum toxin into
subtractions by ion-exchange chromatography in Example 1.
[0022] FIG. 2 is a graphic diagram showing changes in the peak
areas % of subtractions according to changes in the pH of
buffer.
[0023] FIG. 3 is a graphic diagram showing a change in the peak
area % of each subfraction according to the loading volume of a
sample.
[0024] FIG. 4 shows survival rates according to changes in the dose
(A) and time after administration (B) of active fractions (pI and
pII) among the subtractions separated in Example 1 [Mouse: ICR,
female, 4 week-old, 18-22 g, n=10, IM injection into right hind
leg].
[0025] FIG. 5 is a set of photographs showing a comparison of
diffusion to the body from administration site measured at 12 hours
after a non-spreading active fraction (pII) among the subtractions
separated in Example 1 and a commercially available product were
administered into the right hind limbs of mice.
[0026] FIG. 6 is a set of graphs showing survival rates according
to dose (A) and time after administration (B), measured after a
non-spreading active fraction (pII) among the subtractions
separated in Example 1 and a commercially available product were
administered into the right hind limbs of mice.
[0027] FIG. 7 shows a comparison of the time of initiation of
muscle paralysis, measured after 1 U of each of a non-spreading
active fraction (pII) among the subfractions separated in Example 1
and a commercially available product were administered into the
right hind limbs of mice.
[0028] FIG. 8 shows a comparison of the lasting time of muscle
paralysis, measured after 0.5 U of each of a non-spreading active
fraction (pII) among the subfractions separated in Example 1 and a
commercially available product were administered into the right
hind limbs of mice.
BEST MODE FOR CARRYING OUT THE INVENTION
[0029] Hereinafter, each step of a method for purifying a
non-spreading botulinum toxin according to the present invention
will be described in further detail.
[0030] A method for purifying a non-spreading botulinum toxin
according to the present invention comprises the steps of:
separating the botulinum toxin type A product into subfractions
conducting ion-exchange chromatography using buffer of pH 4.5-5.5,
using 0.02-0.2M of sodium chloride (NaCl); and collecting a
non-spreading botulinum toxin subfraction, which has an A260/A280
value of about 0.4-0.6.
[0031] The inventive method for purifying a non-spreading botulinum
toxin is a method wherein an existing botulinum toxin obtained by
either acid precipitation or ion-exchange chromatography following
acid precipitation is subjected to a novel ion-exchange
chromatographic method developed by the present inventors, thereby
obtaining a non-spreading botulinum toxin fraction.
[0032] Thus, the inventive method for purifying a non-spreading
botulinum toxin is performed using a botulinum toxin type A
obtained by either acid precipitation from a culture broth of a
Clostridium botulinum type A strain or chromatographic purification
after acid precipitation.
[0033] When the purified botulinum toxin A subjected to
ion-exchange chromatography using sodium chloride, three
subfractions can be obtained from the ion-exchange resin. To
perform the ion-exchange chromatography, sodium acetate buffer is
used, and the concentration of sodium chloride that is added to the
buffer in order to obtain gradually increased subfractions, and
each separated fraction is taken. It was found that, when no sodium
chloride was used (0 M of NaCl), the fraction separated was an
active fraction (peak I, pI) which spreads in the body, and when
sodium chloride was used at a concentration of 0.02-0.2 M, the
fraction separated was an active fraction (peak II, PII) which does
not spread in the body, and when sodium was used at a concentration
of 1 M, the fraction separated was an inactive fraction (peak III,
pIII) which spreads in the body.
[0034] Specifically, the concentration of sodium chloride that is
used to obtain fraction pII is in the range of 0.02 to 0.2 M, and
when no sodium chloride is used or the concentration of sodium
chloride is out of the above range, fraction pII can contain the pI
or pIII fraction, suggesting that it is difficult to obtain a
non-spreading active fraction having a local muscle-paralyzing
effect sought by the present invention.
[0035] Meanwhile, when the ion-exchange chromatography is performed
using sodium chloride in a buffer having a pH ranging from 4.5 to
5.5, the amount of the pI or pIII fraction in the separated
subfractions is reduced, and the purity of the pII fraction
increases. This can be confirmed by comparing the A260/A280 values
or measuring the degree of paralysis upon injection into the right
hind limb muscles of mice.
[0036] Moreover, when the ion-exchange chromatography is performed
using sodium chloride such that the amount of a purified botulinum
toxin type A sample is 1/5-1 time the volume of the ion-exchange
chromatography column used, the amount of the pII fraction
increases. Specifically, when the column volume is 1 ml and the
amount of the purified botulinum toxin type A sample is 0.2-1 ml,
the amount of the pII fraction obtained is in the range of 20% to
50%.
[0037] Meanwhile, it was found that the pII fraction obtained by
the inventive method for purifying the non-spreading botulinum
toxin type A has a total LD.sub.50 of
1.times.10.sup.5-5.times.10.sup.5 U/ml, as measured using mice. In
addition, it can be seen that the pII fraction does not spread to
areas other than a desired area upon injection and it initiates
muscle paralysis within a significantly short time and maintains
muscle paralysis for a long time compared to a commercially
available product.
[0038] In addition, the pII fraction obtained by the inventive
method for purifying the non-spreading botulinum toxin comprises,
per 100 U,/ml, at least 150 ppb of Zn ions, at least 80 ppb of Fe
ions and at least 140 ppb of Mg ions.
[0039] The inventive method for purifying the non-spreading
botulinum toxin is characterized in that the contents of Mg, Fe and
Zn are significantly higher than those of a botulinum toxin type A
toxin purified by a conventional method.
[0040] It was found that, when the non-spreading botulinum toxin
obtained by the purification method of the present invention was
injected into the right hind limbs of mice in an amount equivalent
to 1.5-3 times the LD.sub.50 of the toxin, the right hind limb
muscles were paralyzed, whereas it did not paralyze respiratory
muscles and had no lethal activity, suggesting that the
non-spreading botulinum toxin of the present invention does not
spread in the body.
MODE FOR CARRYING OUT THE INVENTION
[0041] Hereinafter, the present invention will be described in
detail with reference to examples and the accompanying drawings,
but the scope of the present invention is not limited to these
examples and drawings.
[0042] A commercially available product used in the examples of the
present invention is a Botox product (Allergan, Inc.) prepared by
the method disclosed in U.S. Pat. No. 7,354,740 [entitled "Animal
product free system and process for purifying a botulinum toxin"].
However, the present invention is applied not only to the above
commercially available product or botulinum toxin type A products
purified by the above method, but also to any botulinum toxin type
A product purified by acid precipitation.
Example 1
Fractionation by Ion-Exchange Chromatography
[0043] A sample purified from a botulinum toxin culture medium by
acid precipitation was subjected to ion-exchange chromatography,
thereby separating the sample into three subfractions. The results
of the chromatography are shown in FIG. 1 [pI, peak I (an active
fraction that spreads in the body); pII, peak II (an active
fraction that does not spreads in the body); pIII, peak III (an
inactive fraction)].
[0044] The ion-exchange chromatography was performed using an AKTA
FPLC instrument (GE Healthcare), a Hitrap DEAE FF column (GE
healthcare), and sodium acetate buffer (pH 5.5) as running buffer
(elution buffer).
[0045] Specifically, the sample was eluted with 0 M NaCl (pH 5.5)
in sodium acetate to obtain an unbound wash fraction (pI, an active
fraction that spreads in the body), and then eluted 0.05 M NaCl to
obtain a fraction (pII, an active fraction that does not spread in
the body), and eluted with 1M NaCl to obtain a fraction (pIII, an
inert fraction). The sample was subjected to acid precipitation at
a flow rate of 1 ml/min, followed by desalting.
Example 2
Changes in Peaks and Characteristics as a Function of Changes in
the pH of Buffer
[0046] FIG. 2 shows the peak areas % of the subfractions according
to the changes in the pH of the buffer used in the ion-exchange
chromatography. As can be seen in FIG. 2, when the pH of the buffer
was in the range of 4.5 to 5.5, the peak area % of the pII fraction
increased. Table 1 below shows the A260/A280 values the
subfractions as a function of the pH of the buffer, and Table 2
below shows the total LD.sub.50 (unit) of the subfractions.
TABLE-US-00001 TABLE 1 A260/A280 pH 4.5 pH 5.0 pH 5.5 pH 6.0 pI
0.76 .+-. 0.15 0.69 .+-. 0.09 0.67 .+-. 0.05 0.63 .+-. 0.08 pII
0.50 .+-. 0.05 0.55 .+-. 0.04 0.55 .+-. 0.03 0.81 .+-. 0.19 pIII
0.76 .+-. 0.10 0.82 .+-. 0.13 0.84 .+-. 0.12 0.79 .+-. 0.09
[0047] As can be seen in the above Table 1, when the pH of the
buffer was in the range of 4.5 to 5.5, the A260/A280 value of the
pII fraction was in the range of 0.43 to 0.6, suggesting that the
pII fraction is suitable for administration.
[0048] Thus, it is preferable to adjust the pH of the buffer to
4.5-5.5 in order to obtain a purified active fraction (pII) that
does not spread in the body.
TABLE-US-00002 TABLE 2 Total LD.sub.50 (Unit) pH 4.5 pH 5.0 pH 5.5
pH 6.0 Conventional product 5 .+-. 0.8 5 .+-. 1 5 .+-. 0.8 5 .+-.
1.2 (pre-sample) purified by acid precipitation pI 1.5 .+-. 0.4 3
.+-. 0.5 2 .+-. 0.4 2 .+-. 0.8 pII 0.2 .+-. 0.2 1 .+-. 0.2 3 .+-.
0.4 0.4 .+-. 0.2 pIII -- 0.2 .+-. 0.1 0.2 .+-. 0.1 2 .+-. 1 (Unit:
10.sup.5 U)
[0049] As can be seen in the above Table 2, when the pH of the
buffer was in the range of 5.0 to 5.5, the total LD.sub.50 of the
pII fraction was in the range of 0.8.times.10.sup.5 to
3.5.times.10.sup.5 U/ml, suggesting that return rate of pII is
relatively high.
[0050] Thus, it is preferable to adjust the pH of the buffer to
4.5-5.5 in order to obtain a safe active fraction (pII) that does
not spread in the body.
Example 3
Changes in Peaks and Characteristics as a Function of Changes in
Sample Loading Volume
[0051] Subfractions were obtained in the same manner as in Example
1. FIG. 3 shows changes in the peak area % of the subfractions (pI,
pII and pIII) as a function of changes in the loading volume of the
sample used to obtain the subfractions. Also, Table 3 shows changes
in the A260/A280 values of the subfractions as a function of the
sample loading volume, and Table 4 below shows changes in the total
LD.sub.50 (unit) of the subfractions as a function of the sample
loading volume.
TABLE-US-00003 TABLE 3 A260/A280 0.25 ml 0.5 ml 1 ml 2 ml pI 0.67
.+-. 0.08 0.64 .+-. 0.07 0.68 .+-. 0.09 0.67 .+-. 0.13 pII 0.55
.+-. 0.02 0.56 .+-. 0.01 0.55 .+-. 0.04 0.55 .+-. 0.03 pIII 0.85
.+-. 0.14 0.84 .+-. 0.09 0.82 .+-. 0.18 0.84 .+-. 0.24
[0052] As can be seen in the above Table 3, when the sample loading
volume was in the range of was 0.25 to 2 ml, the A260/A280 of the
pII fraction was 0.4-0.6, suggesting that it is adjustable sample
for administration.
[0053] Thus, it is preferable to control the sample loading volume
in the range of 1/5 to 2 times the column volume in order to obtain
a large amount of an active fraction (pII) that does not spread in
the body.
TABLE-US-00004 TABLE 4 Total LD.sub.50 (unit) 0.25 ml 0.5 ml 1 ml 2
ml Conventional product 2.5 .+-. 0.5 5 .+-. 1 10 .+-. 4 20 .+-. 5
(pre-sample) purified by acid precipitation pI 0.8 .+-. 0.2 1.6
.+-. 0.4 5 .+-. 2 15 .+-. 4.8 pII 1.6 .+-. 0.2 4 .+-. 0.6 4 .+-.
0.9 4 .+-. 1.5 pIII 0.2 .+-. 0.1 0.4 .+-. 0.1 0.8 .+-. 0.2 0.6 .+-.
0.3 (Unit: 10.sup.5 U)
[0054] As can be seen in the above Table 4, when the sample loading
volume was in the range of 0.25 to 1 ml, the total LD.sub.50 of the
pII fraction was in the range of 0.8.times.10.sup.5 to
3.5.times.10.sup.5 U/ml, suggesting that pII return rate is
relatively high.
[0055] Thus, it is preferable to control the sample loading volume
in the range of 1/5 to 2 times the column volume in order to obtain
a safe active fraction (pII) that does not spread in the body.
Test Example 1
Comparison of Muscle-Paralyzing Effects
[0056] 1) Injection into Right Hind Limb Muscles
[0057] In order to examine the muscle-paralyzing effects of the
subfractions obtained in Example 1, injection into the right hind
limb muscles of mice was performed.
[0058] ICR mice (female, 4 week-old, 18-22 g) were divided into
three groups, each group consisting of 10 mice. The weight of each
mouse was precisely measured and recorded. Each of the subfractions
was filled into a 50 .mu.l Hamilton syringe at a concentration of 1
U/20 .mu.l in the absence of air, and then the syringe needle was
pricked into the right hind leg ankle of each ICR mouse to a depth
of about 3 cm, and each of the subfractions was injected into the
muscles of each mouse. After injection into the muscles, limb
paralysis score for each mouse was evaluated at various time points
according to the criteria shown in Table 5, and the survival rate
of the mice was measured. The results of the measurement are shown
in FIG. 4.
TABLE-US-00005 TABLE 5 Score Criteria 0 Limb appearance and walking
of normal mice 1 Mice walk with dragging, but toes are not put
together. 2 Together with criterion of 1, toes are put together. 3
Together with the criterion of 2, foot joints are bent inward. 4
Together with the criterion of 3, feet come into contact with leg
muscles, and legs are lame.
TABLE-US-00006 TABLE 6 Fractions Area paralyzed upon injection into
right hind limbs of mice Active fraction that Various areas in the
body, spreads in the body including right hind limb, diaphragm,
etc. Active fraction that does Right hind limb not spread in the
body Inactive fraction that -- spreads in the body
[0059] As can be seen in FIG. 4, among the subfractions obtained in
Example 1, the active fraction (pII) that does not spread in the
body showed a significant increase in the dose versus survival rate
compared to the active fraction (pI) that spreads in the body (FIG.
4A), and the survival rate at a dose of 2 U was 100% for the pII
fraction and 0% for the pI fraction, which extremely differ from
each other (FIG. 4B).
Test Example 2
Comparison of Diffusion to the Body Between Commercial Product and
Active Fraction (pII) that Does Not Spread in the Body
[0060] In order to examine the muscle-paralyzing effects of a
commercial product (Allergan's Botox) and the subfraction pit
obtained in Example 1, injection into the right hind limb muscles
of mice was performed.
[0061] ICR mice (female, 4 week-old, 18-22 g) were divided into two
groups, each group consisting of 10 mice. The weight of each mouse
was precisely measured and recorded. Each of the commercial product
and the subfraction pII was filled into a 50 .mu.l Hamilton syringe
at a concentration of 1 U/20 .mu.l in the absence of air, and then
the syringe needle was pricked into the right hind leg ankle of
each ICR mouse to a depth of about 3 cm, and each of the commercial
product and the subfraction was injected into the muscles of each
mouse. After injection into the muscles, the degree of paralysis of
the mice was visually observed in order to determine the in vivo
spread of the commercial product and the subfraction. FIG. 5 shows
the state of the mice photographed at 12 hours after injection, and
FIG. 6 shows the survival rate of mice as a function of dose and
administration time. In addition, FIG. 7 shows the time of
initiation of the muscle-paralyzing effect, and FIG. 8 shows the
lasting time of the muscle-paralyzing effect.
[0062] As can be seen in FIG. 5, the subfraction pII paralyzed only
the right hind limb into which it was intramuscularly injected, but
the commercial product did spread to and paralyzed the waist and
the opposite limb.
[0063] As can be seen in FIG. 6, the subfraction pII showed a
significant increase in the dose versus survival rate compared to
the commercial product (FIG. 6A), and the survival rate at a dose
of 2 U was 100% for the pII subfraction and 0% for the commercial
product, which extremely differ from each other (FIG. 6B).
[0064] As can be seen in FIG. 7, the pII subfraction initiated the
muscle-paralyzing effect at a dose of 1 U within a significantly
short time compared to the commercial product.
[0065] As can be seen in FIG. 8, the lasting time of the
muscle-paralyzing effect of the pII subfraction was longer than
that of the commercial product by 80 days or more, at a dose of 0.5
U.
Test Example 3
Analysis of Ion Contents of Freeze-Dried Botulinum Toxins
[0066] A commercial product (Allergan's Botox) and the subfraction
pII obtained in Example 1 were freeze-dried and the ion contents
thereof were analyzed.
[0067] For comparison with the commercial product, the diluted pII
fraction and the same amount of botulinum toxin (100 U) were
freeze-dried with additives, and a mouse lethal test was used to
confirm the freeze-dried products of the commercial product and the
pII subfraction had the same activity, after which the ion contents
of the freeze-dried products were analyzed. Specifically, each of
the two freeze-dried products was dissolved in 10 ml of distilled
water, and then the ion contents thereof were measured using an
inductively coupled plasma mass spectrometer. The results of the
measurement are shown in Table 7 below.
TABLE-US-00007 TABLE 7 Mg Fe Zn pII 183.0 .+-. 19.3* 105.4 .+-.
22.1** 332.6 .+-. 140.8*** Commercial 78.1 .+-. 7.4* 39.6 .+-. 3.3
65.6 .+-. 25.9 product *p < 0.0015, **p < 0.011, ***p <
0.045
[0068] As can be seen in the above Table 7, the contents of Mg, Fe
and Zn in the freeze-dried product of the non-spreading botulinum
toxin (pII fraction) purified by the present invention were
significantly higher than those in the commercial product.
[0069] Although the preferred embodiments of the present invention
have been described for illustrative purposes, those skilled in the
art will appreciate that various modifications, additions and
substitutions are possible, without departing from the scope and
spirit of the invention as disclosed in the accompanying
claims.
Industrial Applicability
[0070] According to the present invention, a non-spreading
botulinum toxin that causes local muscle paralysis can be obtained
in large amounts. This non-spreading botulinum toxin causes muscle
paralysis in a desired area, does not spread to areas other than
the desired area and has the effect of paralyzing muscles in a fast
and lasting manner.
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